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ALLELOPATHIC POTENTIAL OF CONYZA BONARIENSIS
By
MATSELENG WENDY MALATJI
04283775
Submitted in partial fulfillment of the requirements for the degree
M Inst Agrar Agronomy
in the Department of Plant Production and Soil Science, Faculty of Natural and
Agricultural Sciences,
University of Pretoria
Supervisor: Prof. C.F. Reinhardt
Co-supervisor: Dr. N.J. Taylor
November 2013
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© University of Pretoria
DECLARATION
I, the undersigned, hereby declare that the dissertation submitted herewith for the
degree M Inst Agrar Agronomy to the University of Pretoria, contains my own
independent work and has not been submitted for any degree at any other university.
_____________________
Matseleng W. Malatji
November 2013
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Allelopathic potential of Conyza bonariensis
by
Matseleng Wendy Malatji
Submitted in partial fulfilment of the requirements
for the degree M Inst Agrar Agronomy
In the Department of Plant Production and Soil Science
Faculty of Natural and Agricultural Sciences
University of Pretoria
PRETORIA
Supervisor:
Co-supervisor:
Prof. C. F. Reinhardt
Dr. N. J. Taylor
ABSTRACT
Conyza bonariensis, flaxleaf fleabane, is a major weed threat on cultivated and noncultivated lands, gardens, roadsides and waste places. The weed in South Africa is
believed to have originated from South America, and the first herbarium sample is
from a plant collected in May 1895 at Franschhoek. Adding to its problem status is
the recent discovery that certain C. bonariensis biotypes in South Africa and other
parts of the world are resistant to the herbicide glyphosate, and in certain cases to
both glyphosate and paraquat. Despite its invasiveness and ability to compete
severely with crops, the mechanisms of interference (= allelopathy + competition)
employed by C. bonariensis are poorly understood and have not yet been thoroughly
investigated. There is a need to expand on the knowledge of interference
mechanisms of C. bonariensis in order to better understand its success as a weed,
and to improve on knowledge for the successful management of this weed. In the
present study, allelopathic potential of C. bonariensis was assessed, first by means
of germination bioassays, followed by investigation employing hydroponics, leachate,
and replacement series experiments. In a laboratory bioassay, the plant’s leaves and
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roots were extracted using two solvents, water and hexane, to which seeds of the
test (acceptor) species lettuce (Lactuca sativa L.) and tomato (Lycopersicon
esculentum) were exposed in order to determine where the strongest allelopathic
potential resides. Moreover, differential potency of crude extracts prepared with the
two solvents (polar and non-polar) would at least provide some evidence on the
nature of putative allelochemicals involved. Germination bioassays revealed that
leaves harboured the strongest allelopathic potential (potency). Water extracts
(infusions) caused greater growth inhibition of the test species than hexane extracts.
Osmolalities of the water infusions were tested and found not to be inhibitory to
germination and early seedling development of lettuce. Following on the germination
bioassays, a hydroponic experiment was set up in a greenhouse in order to
investigate whether C. bonariensis possesses and releases chemicals with
allelopathic potential through its roots. Lettuce top and root growth was significantly
reduced by all three populations of C. bonariensis (one from Pretoria; two from the
Western Cape). No significant differences were observed in the degree of growth
inhibition caused by the three weed populations on the growth of lettuce, except in
the case of root dry mass results where the Hatfield population caused more
damage (85% growth reduction). The leachate experiment was then performed to
determine if leachate from C. bonariensis affected the growth of test species
exposed to different leachate concentrations. Although there was no growth
inhibition observed for both lettuce and tomato in this experiment, growth stimulation
of tomato roots was observed at the highest leachate concentration (100%). Finally,
in an attempt to simulate the allelopathic potential of C. bonariensis in a natural field
situation, a replacement series experiment was conducted to determine the relative
interference of Conyza bonariensis in relation to lettuce and tomato. Dry mass
results showed that there was no growth inhibition of both crop species. RYT was >
1 at all weed: crop combinations, which implies that both crop species and C.
bonariensis were less affected by interspecific interactions than in their respective
monocultures. It is suggested that the results of this study can attributed to
methodology and growth media. The results of this study represent the first step in
showing that allelopathic potential C. bonariensis may contribute to the success of
this weed as an invasive weed species and that this weed should not be allowed to
attain significant biomass on crop field. Further research should include field trials
that will yield a better understanding of the practical relevance of the allelopathic
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potential of C. bonariensis. Finally, crop producers and weed management
practitioners should recognize that this important weed has the ability to interfere
with the growth and development of a crop through two mechanisms, competition
plus allelopathy.
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ACKNOWLEDGEMENTS
I dedicate this study to my mother, Khomotso Malatji, thank you for believing in me. I
will be eternally grateful for the sacrifices and the support you gave me to complete
this study. You were the light and driving force.
I acknowledge with appreciation my indebtedness to Professor C.F. Reinhardt
for the support, patience and generous guidance provided during the
conceptualisation and implementation of this study.
Dr Nicolette Taylor her incredible insight and much appreciated help.
Mr. Ronnie Gilfillan for his assistance and support with lab work.
Mr. Jacques Marneweck and the staff from the Hatfield experimental farm for
their assistance throughout the trial phase.
Tsedal Tseggai Ghebremariam for her help with the statistical analysis.
Department of Plant Production and Soil Science and Plant Sciences for the
use of their facilities.
National Research Foundation and Monsanto for their financial support.
My siblings Shadi, Mpho, Thabo, your support and inspiration are a priceless
gift. Thank you for your encouragement.
To
Desiree,
Lethabo,
Edwin,
and
Kanyo,
without
your
emotional
encouragement and unfailing friendship, there would have been no final
dissertation. Thank you for being so sincere…
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CONTENTS
Declaration
i
Abstract
ii
Acknowledgements
v
List of abbreviations
ix
List of tables
x
List of figures
xi
Introduction
1
CHAPTER 1
Literature review
1.1 Invasive alien plants
1.2 Allelopathy: A background
1.2.1 Brief definition and history
1.2.2 Interactions of allelochemicals
1.2.3 Allelopathy and agriculture
1.2.4 Allelopathy and biodiversity
1.2.5 Assessing allelopathic potential
1.3 Conyza species
1.3.1 Botanical description
1.3.2 Distribution and habitat of Conyza spp in South Africa
1.3.3 Interference and allelopathic potential of Conyza
species
1.3.4 Control measures
CHAPTER 2
3
5
6
7
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10
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19
21
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Allelopathic influence of Conyza bonariensis on lettuce
and tomato seed germination and early seedling
development
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CHAPTER 3
CHAPTER 4
CHAPTER 5
2.1 Introduction
27
2.2 Materials and Methods
29
2.2.1 The bioassay technique
29
2.2.2 Exclusion of osmotic potential effects
31
2.3 Results and Discussion
34
2.4 Conclusion
46
Assessment of the allelopathic potential of Conyza
bonariensis root exudates
3.1 Introduction
47
3.2 Materials and Methods
49
3.2.1 Hydroponic experiments
49
3.2.2 Leachate experiment
51
3.3 Results and Discussion
54
3.4 Conclusion
62
Replacement series approach for determining the
relative interference of Conyza bonariensis in relation
to lettuce and tomato
4.1 Introduction
64
4.2 Materials and Methods
66
4.3 Results and Discussion
69
4.4 Conclusion
75
GENERAL DISCUSSION AND CONCLUSION
76
SUMMARY
80
REFERENCES
82
APPENDIX A
100
APPENDIX B
104
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APPENDIX C
109
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LIST OF ABBREVIATIONS
ANOVA
: Analysis of variance
gL-1
: grams per litre
LSD
: Least significant difference
mOsm kg-1
: milliOsmol per kilogram
PEG
: polyethylene glycol
RY
: relative yield
RYT
: relative yield total
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LIST OF TABLES
Table 1.1
Herbicides registered to control Conyza species
Table 2.1
The effect of PEG-6000 solutions of increasing osmolality on
22
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germination and radicle and shoot lengths of lettuce seedlings
Table 2.2
The effect of PEG-6000 solutions of increasing osmolality on
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germination and mean radicle and shoot length of lettuce seedlings
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LIST OF FIGURES
Figure 1.1
Conyza sumatrensis
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Figure 1.2
Conyza bonariensis
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Figure 1.3
Conyza canadensis
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Figure 1.4
A map showing the distribution of glyphosate and paraquat resistant
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C. bonariensis in the Western Cape, South Africa
Figure 2.1
Effect of aqueous leaf and root extracts of C. bonariensis on seed
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germination of lettuce
Figure 2.2
Effect of aqueous leaf and root extracts of C. bonariensis on root
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(radicle) growth of lettuce
Figure 2.3
Effect of aqueous leaf and root extracts of C. bonariensis on shoot
growth of lettuce
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Figure 2.4
Effect of aqueous leaf and root extracts of C. bonariensis on seed
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germination of tomato
Figure 2.5
Effect of aqueous leaf and root extracts of C. bonariensis on root
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growth (radicle) of tomato
Figure 2.6
Effect of aqueous leaf and root extracts of C. bonariensis on shoot
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growth of tomato
Figure 2.7
Effect of hexane leaf and root extracts of C. bonariensis on root
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growth of lettuce
Figure 2.8
Effect of hexane leaf and root extracts of C. bonariensis on root
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(radicle) growth of lettuce
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Figure 2.9
Effect of hexane leaf and root extracts of C. bonariensis on shoot
43
growth of lettuce
Figure 2.10
Effect of hexane leaf and root extracts of C. bonariensis on seed
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germination of tomato
Figure 2.11
Effect of hexane leaf and root extracts of C. bonariensis on root
45
(radicle) growth of tomato
Figure 2.12
Effect of hexane leaf and root extracts of C. bonariensis on shoot
45
growth of tomato
Figure 3.1
Hydroponic system used to study the effect of allelochemicals
49
released by the roots of C. bonariensis plants on lettuce seedlings
Figure 3.2
Hydroponic system in which one C. bonariensis from either
51
Naboomsrivier or Willow Creek Boerdery were grown with two
lettuce seedlings; C. bonariensis and lettuce plants grown on their
own served as control
Figure 3.3
Test species that were used in the C. bonariensis leachate
experiment: lettuce seedlings and tomato seedlings
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Figure 3.4
Leachate experiment for the assessment of allelopathic effects of C.
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bonariensis; Mitscherlich pots with C. bonariensis plants (donor
plants) were supplied with pans at bottom for leachate collection
Figure 3.5
Shoot and root fresh mass of test species lettuce grown
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hydroponically with C. bonariensis plants collected on the Hatfield
experimental farm
Figure 3.6
Root growth variation between the roots of lettuce grown alone and
lettuce grown with C. bonariensis
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Figure 3.7
Root and shoot mass comparison between plants representing the
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controls of C. bonariensis and lettuce (left side of ruler), and plants
from the weed-crop combination treatment (right side of ruler)
Figure 3.8
Shoot and root dry mass of
test species lettuce grown
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hydroponically with C. bonariensis plants collected on the Hatfield
experimental farm
Figure 3.9
Shoot and root fresh mass of test species lettuce grown
hydroponically with two Western Cape provenances of
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C.
bonariensis
Figure 3.10
Shoot and root dry mass of
test species lettuce grown
hydroponically with two Western Cape provenances of
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C.
bonariensis
Figure 3.11
Shoot and root fresh mass of lettuce that was exposed to C.
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bonariensis leachate concentrations ranging from 0 to 100%
Figure 3.12
Shoot and root dry mass of lettuce plants exposed C. bonariensis
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leachate concentrations ranging from 0 to 100%
Figure 3.13
Shoot and root fresh mass of tomato plants exposed to different C.
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bonariensis leachate concentrations ranging from 0 to 100%
Figure 3.14
A: Roots of tomato grown in pure nutrient solution; B: roots of plants
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treated with 100% C. bonariensis leachate concentration
Figure 3.15
Shoot tops and root dry mass of tomato plants exposed to a range
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of C. bonariensis ranging from 0 to 100%
Figure 4.1
A replacement series experiment to investigate the effect of different
densities of C. bonariensis on plant growth of lettuce
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Figure 4.2
Dry mass of C. bonariensis and lettuce grown together in a
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replacement series at different proportions.
Figure 4.3
Root and shoot growth comparison between C. bonariensis and L.
sativa from the replacement series. A: 5 lettuce + 0 C. bonariensis;
B: 5 C. bonariensis + 0 lettuce; C: 4 C. bonariensis +1 lettuce; D: 3
C. bonariensis +2 lettuce; E: 2 C. bonariensis + 3 lettuce; F: 1 C.
bonariensis + 4 lettuce
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Figure 4.4
Dry mass of Conyza bonariensis and tomato grown together in a
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replacement series at different proportions
Figure 4.5
Root and shoot growth comparison between C. bonariensis and
tomato from the replacement series. A: 5 tomato + 0 C. bonariensis;
B: 5 C.bonariensis + 0 tomato; C: 4 C. bonariensis + 1 tomato; D: 3
C. bonariensis + 2 tomato; E: 2 C. bonariensis + 3 tomato; F: 1 C.
bonariensis + 4 tomato
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Figure 4.6
Relative yields (RY) of lettuce and C. bonariensis and relative yield
74
total (RYT) four weeks after transplanting under different densities
and proportions
Figure 4.7
Relative yields (RY) of tomato and C. bonariensis and relative yield
total (RYT) four weeks after transplanting under different densities
and proportions
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75
INTRODUCTION
The invasion of newly colonised areas by alien species is a problem of great
significance globally. Apart from displacing the indigenous plants, these plants are
able to survive, reproduce and spread at alarming rates. The comprehension of
survival mechanisms utilized by such species is an imperative process before
implementing control strategies. Conyza spp among many other invasive alien
species have become major weed pests in South Africa (Bromilow, 2010) and other
parts of the world (Heap, 2012). Although the first record of the Conyza spp in the
country was over a century ago, they seem to have become more troublesome in
recent years.
While all plant species compete to survive, invasive species appear to have specific
traits or a combination of these traits, which allow them to out compete native
species (Kolar and Lodge, 2001).Facilitation is the mechanism that some species
use to change their environment through chemical or physical manipulation of biotic
and abiotic factors, usually to make conditions unfavourable for other species which
compete with them. Allelopathy is an example of a chemical facilitative mechanism
(Hierro and Callaway, 2003). Among the weed species reported worldwide a
considerable number reportedly possess allelopathic potential. In allelopathic
interactions there is production and release of chemical substances by certain plants
aimed at inhibiting the growth and development of neighbouring species. They are
released into the environment by root exudation, leaching from aboveground parts,
and volatilisation and/or by decomposition of plant material, and can be present in
several parts of plants including roots, rhizomes, leaves, stems, pollen, seeds and
flowers. Several papers have suggested allelopathy as an alternative to weed
management (Macias, 1995; An et al., 1998; Inderjit and Keating, 1999). Options
such as using allelochemicals as herbicides, and improving the allelopathic activity of
crops through breeding strategies or by genetic engineering have been explored
(Macias, 1995; Chou, 1999).
In South Africa there are three main species of Conyza namely Conyza canadensis,
Conyza bonariensis, Conyza sumatrensis, commonly known as Canadian fleabane,
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flax-leaf fleabane, and tall fleabane respectively. Of the three, C. bonariensis and C.
sumatrensis seem to have a wide distribution in the country. Previously the biology
and ecology of Conyza have been the main focus of studies, which entailed studies
on population dynamics, seed production, emergence and distribution. Other studies
on the weed focused on the resistance of C. bonariensis to herbicides, and it being
the first broadleaf weed documented as resistant to glyphosate (Shrestha and
Hembree, 2005; Heap, 2006; Weaver, 2001). Conyza spp have succeeded as wellequipped competitors in a range of habitats and ecosystems. With the exception of
C. canadensis and C. sumatrensis, little attention has been given to the competitive
advantages that aid Conyza spp in survival to persist in new environments and
foreign lands.
The main aim of the study was to determine if C. bonariensis in South Africa possess
allelopathic potential, specifically the ability to suppress crop growth, through the
release of allelochemicals from the roots. The hypothesis is thus that C. bonariensis
produces compounds with allelopathic potential that affect the growth of surrounding
plants, thereby gaining a competitive advantage.
Specific objectives were the following:
To verify whether C. bonariensis has different impacts on seed germination
and seedling growth of a test species;
To evaluate the influence of different plant parts of C. bonariensis on seed
germination and seedling growth of the test species;
To investigate whether C. bonariensis possess chemicals with allelopathic
potential by growing it together with test species in a nutrient solution and
using plant growth as measure of effect;
To verify if different biotypes of C. bonariensis would have the same effect on
the growth of the same test species;
To determine test plant responses to different concentrations of root leachate
collected from C. bonariensis plants;
To assess the interference of C. bonariensis with growth of the test species by
increasing C. bonariensis plant density, and thus the concentration of
compounds with allelopathic potential in the growth medium.
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CHAPTER 1
LITERATURE REVIEW
1.1 Invasive alien plants
A great number of plant species have the ability to grow in conditions that are similar
but also quite different from those in their native habitats. Consequently, many plants
are currently in places where they never existed before. The term invasive species
refers to non-indigenous species that affect the habitats they invade environmentally,
ecologically, and economically (Kolar and Lodge, 2001). These types of plants are
able to survive, reproduce and spread unaided sometimes at alarming rates across
the landscape. Their impact on agriculture is considered to be significantly greater in
developing than in developed countries (Perrings, 2005).
In a review on the impact and management of invasive plants in Africa, Witt (2010)
states that, the impact of invasive alien species on the continent, especially
introduced weeds, is significant because more than 80% of the population are smallscale farmers who are dependent on natural resources for their survival. In countries
such as Angola, Zambia, Malawi, Uganda, Mozambique, Ethiopia, and Sudan the
agricultural labour force is about 80% of the total labour force, and hand weeding
accounts for up to 60% of pre-harvest labour (Webb and Conroy, 1995; Witt, 2010).
In South Africa it has been documented that thousands of plant species have been
brought into the country for a range of purposes, such as crop species for timber and
firewood, as garden ornamentals, for stabilizing sand dunes as barriers and hedge
plants (Van Wilgen et al., 2001). An estimated 750 tree species and around 8000
shrubby succulent and herbaceous species have been introduced to South Africa,
with 161 species regarded as seriously invasive. Suggestions that 750 000 ha of
invaded land should be cleared annually, if the battle against invasive plants is to be
won within 20 years, have been made (MacDonald et al., 2003). However, this 20year effort would come at a projected cost of R5.5 billion (Le Maitre et al., 2000).
While all plant species compete to survive, invasive species appear to have specific
traits or a combination of these traits, which allow them to out-compete native
species (Kolar and Lodge, 2001). An introduced species might become invasive if it
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can out-compete native species for resources such as nutrients, light, physical
space, and water. Invasive species might be able to use resources unavailable to
native species, such as deep water accessed by a long taproot, or an ability to live
on previously uninhabitable soil types. These species have evolved under great
competition and predation, and the new environment allows them to proliferate
quickly (Stohlgren et al., 1999). Perfect examples of successful invader alien plants
are: Parthenium hysterophorus which is present in Kenya, Uganda, Tanzania, South
Africa, Mozambique, and Swaziland, and is currently considered to be the most
important weed in both croplands and grazing areas by 90% of farmers in the
lowlands of Ethiopia (Tamado and Millberg, 2000), with sorghum yields being
reduced by 97% in experimental fields with high densities of parthenium (Tamado et
al., 2002). The impact of parthenium has also been well documented in Australia and
India (Evans, 1997). Conyza spp are another invader group of weeds that were
introduced into South Africa about a century ago from South and North America.
They now cause problems in cultivated and non-cultivated lands, gardens, roadsides
and waste places (Ciba-Geigy, 1985).
Many exotic plant species competitively exclude and eliminate their neighbours in
invaded ―recipient‖ communities, but coexist in relative harmony with neighbours in
species-diverse systems in their native habitat (Hierro and Callaway, 2003).
Researchers have suggested that this is due to the existence of empty niches in
recipient communities, rapid genetic changes in invader populations in response to
selection pressure in the novel environment, and special adaptation to human
disturbance by invaders (Mack et al., 2000; Sakai et al., 2001).
The primary theory for the unusual success of invasive plants is that they have
escaped the natural enemies that hold them in check, thus freeing them to utilize
their full competitive potential – the ―natural enemies hypothesis‖ (Darwin, 1859;
Williams, 1954; Elton, 1958; Gillet, 1962). The hypothesis has been tested around
the world by releasing hundreds of types of biocontrol agents, but the majority of
them have been ineffective (Maron and Vila, 2001). This indicated that an inquiry
about mechanisms for the general success of many exotic invasive plants was
essential for gaining control over invading species.
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Several mechanisms have been proposed to explain invasive plant species success
within introduced areas compared to their natural ranges. More recently, allelopathy
has been suggested as a potentially important mechanism of plant invasion success,
particularly when the invaders produce evolutionarily novel chemicals (Hierro and
Callaway, 2003; Inderjit et al., 2008).
1.2 Allelopathy: A background
In their communities plants will interact either positively or negatively. However, it is
more common that neighbouring plants will interact in a negative manner, whereby
the emergence and growth of one or more engaged in the interaction, is inhibited.
This adverse effect of a neighbouring plant in an association is termed interference
(Muller, 1969; Foy and Inderjit, 2001). Plant interference is generally explained by
two phenomena, resource competition and allelopathy. Competition implies limitation
of resources such as light, water, space, and nutrients, and allelopathy can be
defined as all effects of plants on neighbouring plants through the release of
chemical compounds into the environment (Rice, 1984).
In nature it is particularly difficult to separate allelopathic interference from resource
competition because there are many factors interacting simultaneously (Weston and
Duke, 2003). Proof of allelopathy involves isolating compounds and demonstrating
that a toxic effect on other plant species is the main function of the compound and
that when the other interactions such as resource limitations are alleviated, the
allelopathic effect persists (Williamson, 1990). Under controlled conditions, factors in
competition may be separated, and it is possible to prove that chemical interactions
are either totally or partially responsible for the interference observed. In devising
laboratory and greenhouse studies, efforts have been made to assure that the
biological activities obtained are indeed due to the extracellular toxins by the donor
plants (Qasem and Foy, 2001). For example, Belz et al. (2009) conducted a study to
investigate whether or not the plant metabolite parthenin is sufficiently persistent,
phytotoxic, and bioavailable in soils to cause an allelopathic effect that makes it
attributable to the invasive success of the weed P. hysterophorus. In this study,
parthenin was found to be quickly degraded without any evident accumulation to
toxic levels over time and therefore; the hypothesis that parthenin contributes to the
invasiveness of P. hysterophorus was rejected.
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Allelopathy has been suggested as a mechanism for the success of invasive plants
by establishing a virtual monoculture and may contribute to the ability of particular
exotic species to become dominant in invaded plant communities (Hierro and
Callaway, 2003). It is expected to be an important mechanism in the plant invasion
process because the lack of co-evolved tolerance of resistant vegetation to
chemicals produced by the invader, which allows the newly arrived species to
dominate natural plant communities.
1.2.1 Brief definition and history
The concept of allelopathy has been cited in literature for over 2000 years (Weston
and Duke, 2003). Theophrastus (372 to 285 BC), a disciple of Aristotle, speculated
that there might be chemical interactions between weeds and plants but provided
little evidence to substantiate this claim. De Candolle (1932), a pioneer in allelopathy
research of weeds on crop plants, concluded that exudates of certain weed species
injured specific crop plants. His research stimulated interest in the chemical ecology
of plants, but it was Molish (1937) who coined the term allelopathy, derived from the
Greek words allelon (of each other) and pathos (to suffer).Rice (1984) defines
allelopathy as any direct or indirect effect by one plant, including micro-organisms,
on another through the production of chemical compounds that escape into the
environment and subsequently influence the growth and development of
neighbouring plants.
Over the last three decades, there has been an increase in publications on
allelopathy and a considerable amount of literature is available that implicates
allelopathy as an important form of plant interference. The term is today generally
accepted to cover both inhibitory and stimulatory effects of one plant on another
plant (Qasem and Foy, 2001).
In 1996 the International Allelopathy Society defined allelopathy as follows: ―Any
process involving secondary metabolites produced by plants, micro-organisms,
viruses, and fungi that influence the growth and development of agricultural and
biological systems (excluding animals), including positive and negative effects‖
(Torres et al., 1996).Ten years ago, 240 weed species were reported to have
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inhibitory action on crop plants alone. Major progress in the science has recently
occurred and the phenomenon is of worldwide importance (Qasem and Foy, 2001).
1.2.2 Interactions of allelochemicals
Allelochemicals cause germination and growth inhibition, and influence a wide
variety of metabolic processes. These substances can be isolated from plant tissues.
Allelochemicals can be found in numerous parts of a plant such as roots, rhizomes,
leaves, stems, pollen, seed, and flowers, and are usually products of secondary
plant metabolism (Rice, 1984).
The most important allelochemicals include alkaloids, terpenoids, flavonoids,
steroids, tannins, and phenolic compounds (Whittaker and Feeny, 1971; Mandava,
1985; Shakaut et al., 2003). Phenolic compounds are reported to constitute the
principal allelopathic agents in weeds and other allelopathic plants. Often their
function in the plant is unknown but some allelochemicals are reported to have
structural functions e.g., as intermediates of lignification or play a role in general
defence against pathogens (Niemeyer, 1988; Corcuera, 1993; Einhellig, 1995).
Allelochemicals are released into the environment by root exudation, leaching from
aboveground parts, and volatilisation and/or by decomposition of plant material
(Rice, 1984), and their ability to persist in soil is determined by sorption, fixation,
leaching and chemical or microbial degradation (Inderjit, 1998). The degree of
phytotoxicity depends on residue persistence and the extent of dissipation in the soil
environment.
According to Inderjit and Weiner (2001) allelochemical effects in the field could be
due to four possibilities: (i) direct harmful effects of chemicals released from donor
plants, (ii) degraded or transformed products of released chemicals, (iii) effect of
released chemicals on physical, chemical and biological soil factors, and (iv)
induction of release of biologically active chemicals by a third species. Since it is
difficult to distinguish between these four possibilities, Inderjit and Weiner (2001)
proposed that allelopathy be understood in its ecological context rather that based
on direct plant-plant allelopathic interference.
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Allelopathy is strongly coupled with other stresses of the crop environment including
insect and disease, temperature extremes, light, nutrients and moisture variables,
and herbicides, and is strongly influenced by habitat ecology (Inderjit and Keating,
1999).Environmental factors also have the ability to influence the production of
allelochemicals and their effects. Plants growing in resource-limited environments
exhibit higher tissue concentration of secondary compounds when compared to
those growing under less stressful conditions. For example, Koeppe (1976) found
that increased amounts of allelopathic substances were produced when plants grew
in phosphorus-deficient soil.
Drought has been reported to have ability to increase the amount of allelopathic
compounds in soil (Gershenzon, 1984).It has been shown that allelopathic activities
are more pronounced when plant species grow underwater stress (Einhellig, 1987,
1989). Ardi (1986) found that the reduction of sweet corn (Zea mays) yield due to
purple nutsedge (Cyperus rotundus) was most severe when the greatest water
stress was imposed. Thus, growth inhibition of sweet corn may be due to the
combined stress of direct water deficit and greater production of allelopathic
substances in purple nutsedge under these conditions.
Chemicals released by plants including allelochemicals also play an important role in
influencing ecological processes in plant communities through their effects on soil
ecology (Wardle et al., 1998). Many secondary metabolites such as phenolics and
terpenoids are known to form complexes with organic ions and influence
accumulation of nutrients. Phenolics may affect phosphate availability by competing
for anion absorption sites. They can bind to Al, Fe, and Mn, thus releasing
phosphate otherwise bound to these cations (Appel, 1993).
Allelochemicals may also influence microbial ecology by their effects on soil
microbes and plant pathogens. Population densities of soil-borne microorganisms
are affected by soil enrichment with phenolic acids, ferullic, p-coumaric, and vanillic
acids (Blum and Shafer, 1988). However, microbial degradation of allelochemicals
may prevent them from reaching phytotoxic levels in natural soils (Schmidt and Ley,
1999). Soil is a very complex system and it affects both the quantitative and
qualitative ability of allelochemicals and therefore allelopathic responses of the plant
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(Inderjit et al., 1999). Inderjit and Weiner (2001) suggested that research on the
influence of allelochemicals on different components of the soil ecosystem and their
role in shaping community structure and composition is needed.
The study of allelopathy therefore has numerous aspects or dimensions, namely:
ecology, plant physiology, microbiology, molecular biology, natural product chemistry
and agriculture. Its application to agricultural production has been anticipated and
researchers have found allelopathic plants that are now used as cover crops for
sources of allelochemicals, and these compounds are serving as leads in the
development of new herbicides (Hirai, 2003).
1.2.3 Allelopathy and agriculture
Weeds account for more than 1% of the total plant species on earth, but cause great
damage by interfering with food production, health, economic stability and welfare
(Qasem and Foy, 2001). They may be defined as plants with little economic value
and possessing the potential to colonize disturbed habitats or those modified by
human activities (Macias et al., 2004). Simply put, weeds are often plants that are
uniquely adapted to a wide range of environmental conditions, and they did not
acquire problem status until humans developed agriculture. Therefore, it is up to
humans to find a solution to the problems weeds cause in agriculture.
Various researchers have referred to allelopathic agents as the future natural
pesticides or nature‘s herbicides in action (Putnam, 1983; Rice, 1995). Qasem and
Foy (2001), state that the limited work on mode of action of allelochemicals suggests
that they affect a variety of sites and biochemical processes, many of which are
familiar to those affected by synthetic herbicides. Allelochemicals are considered
safer than synthetic chemicals because of their biodegradability.
Allelopathic crops, when used as cover crops, mulch, green manures, or grown in
rotation, are helpful in reducing noxious weeds and plant pathogens (Khanh et al.,
2005). Common examples of crops exhibiting allelopathy include, Sorghum bicolor
(Putnam, 1983), Triticum aestivum (Kimber, 1973), Oryza sativa (Chou, 1995) and
Zea mays (Yakle and Cruse, 1984).
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Crop rotation is reported to have a greater effect on weed species and densities than
tillage practices (Weston, 1996), and the practice simultaneously controls pests,
enhances ecosystem diversity and improves crop productivity (Mamolos and
Kalburtji, 2001). Japanese farmers use beans in spring, buckwheat in summer and
then wheat in winter (Kahn et al., 2005). The beans are reported to help with soil
nutrient enrichment, whilst buckwheat is known as a weed ―killer‖ and can be used
as green manure that contributes to soil nutrients. Therefore, buckwheat plants are
incorporated in the soil to help reduce weeds and increase the yield of wheat.
Microorganisms can be considered as a source of new allelochemicals; hence their
phytotoxic and pharmacologic properties have created growing interest (Macias et
al., 2004). According to Khalid et al. (2002), microbially produced phytotoxins have
more potential than some herbicides, because they are selective and, compared to
using the actual pathogens, they are easy to formulate, less likely to spread diseases
to non-target species, and their activity is less dependent on environmental
conditions. This comparison may hold true for certain microbial toxins and synthetic
herbicides, but mostly the latter are more selective in terms of controlling weeds
without harming the crop, and they have better residual activity than most herbicides
of biological origin.
Allelopathy, as a science, is rapidly growing and its significant role in nature is now
fairly well acknowledged. However, more experimental evidence and a great deal of
more intensive, precise investigation is still required (Qasem and Foy, 2001). With
modern analytical technical methods (HPLC, GC-MS, IR, NMR, etc.), more
allelochemicals are likely to be isolated to produce bioactive herbicides and
pesticides (Khan et al., 2005).
1.2.4 Allelopathy and biodiversity
After direct habitat destruction, biological invasions have been viewed as the second
largest global threat to diversity, given their effect on agriculture, forestry and human
health (Wilcove et al., 1998; Walker and Steffen, 1999). It has been suggested by
global reviews that the most harmful species transform ecosystems by utilising
excessive amounts of resources (particularly water, light and oxygen), by adding
resources (particularly nitrogen), by promoting or suppressing fire, by stabilizing
sand movement and/or promoting erosion by accumulating litter or by accumulating
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or redistributing salt (Richardson et al., 2000). According to Vitousek (1990), these
changes possibly alter the flow, availability or quality of nutrient resources in
biogeochemical cycles; they modify trophic resources within food webs; and they
alter physical resources such as living space or habitat, sediment, light and water.
Therefore, alien invaders are likely to act as ‗ecosystem engineers‘ by rapidly
changing disturbance regimes (Crooks, 2002).
The importance of plant diversity is due to its ability to provide insurance against
large changes in ecosystem processes and manage efficiency of resource utilization
(Inderjit and Foy, 2001). Reduction in genetic diversity of crops and wild plants is a
direct consequence of loss in plant diversity (Solbrig, 1991). Through evolutionary
processes both competition and allelopathy play important roles in regulating the
species diversity in a plant community (Inderjit and Foy, 2001). Allelopathic
compounds have been shown to play important roles in determining plant diversity,
dominance, succession and climax of natural vegetation, and in the plant productivity
of agroecosystems (Chou, 1999). By applying an excess of fertilizers, herbicides,
fungicides, and nematacides, etc., modern agricultural practices can jeopardize the
physical-chemical properties of the soil, and pollute the soil and water to the
detriment of the global ecosystem (Chou, 1999). In order to achieve the goal of
sustainable agriculture, extensive research is needed and has been done on plant
breeding, soil fertility and tillage, crop protection, and cropping systems (Chou,
1999).
1.2.5 Assessing allelopathic potential
Bioassays, as a tool for assessing the biological activity of natural and synthetic
chemicals, are defined as the assessment of the potency of a compound via the
application-induced response to that compound (Webster, 1980; Govindarajulu,
1988). In allelopathy, bioassays are necessary in each step of the isolation,
purification, and identification processes of active compounds (Rice, 1974).
Bioassays are an important part of allelopathy studies that employ whole plants,
plant parts or plant tissues. Bioassays have been successful in detecting the
biological activity of several synthetic compounds and natural products (Inderjit and
Nilsen, 2003).
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Allelochemicals produced in nature are largely influenced by habitat ecology and
environmental factors (Inderjit, 1996). Different mechanisms of interference may
occur at the same time, therefore making it difficult to separate these mechanisms at
the field level (Inderjit and Dakshini, 1995). Laboratory, greenhouse, and growth
chamber bioassays provide controlled conditions which allow the researcher to have
control over the interactions that take place in nature. Numerous bioassays have
been proposed for testing allelopathy; however, there also has been criticism on
them often providing little or no connection to plant interactions that occur in the field,
because it is difficult for bioassay experiments to simulate natural field conditions
(May and Ash, 1990). However, the presence of phytotoxic chemicals in a plant does
at least imply allelopathic potential in a natural setting (Heisey, 1990). For definite
proof of allelopathy, demonstration that the allelopathic compound is released into
the environment at a concentration high enough to cause allelopathic effects is
essential (Inderjit and Keating, 1999). The use of various test plant species in
bioassays can provide information on the phytotoxicity, selectivity or species
sensitivity to allelochemicals (Hoagland and Williams, 2003). Specific molecular
assays can also be performed on proven allelochemicals in order to elucidate
modes-of-action (absorption, translocation and mechanism-of-action).
Hoagland and Williams (2003) state that bioassays have inherent limitations, such
as: exhibition of large standard errors for means in dose-response curves compared
to data from physicochemical methods, and the presence of interfering substances in
non-purified extracts that may have greater effects in bioassays than in
physicochemical analyses. They proposed that these limitations can be minimized by
proper experimental designs, test material, test methodology, replication, and
judicious selection of statistical analysis method. Furthermore, they pointed out that
improved techniques such as HPLC, GC, mass spectrometry, NMR, immunological
methods, etc., provide greater sensitivity/specificity and are more accurate than
bioassays.
Among the many measures of phytotoxicity of allelochemicals, the inhibition (or
stimulation) of seed germination, radicle elongation, and/or seedling growth in soil
with surface debris or containing incorporated plant debris, have been the
parameters of choice for most investigations (Leather and Einhellig, 1986). These
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parameters are accepted as indirect measures of other physiological processes
affected by chemical interaction. In this way, a wide range of effects are covered,
and such bioassays serve to select compounds that can be evaluated in greenhouse
and field studies (Macias et al., 2000).
1.2.5.1General plant bioassays
1.2.5.1a Germination bioassays
In general, extract bioassays are conducted in Petri dishes, by placing seeds of the
receiver species on substrate (often filter paper) moistened with aqueous plant
extracts of donor species (Wu et al., 1998). During extraction, care should be taken
to ensure that seed germination is not delayed by the osmotic potential of the extract
solution (Hoagland and Williams, 2003). The Petri dishes are placed in an incubator
under controlled light and dark periods, and are regularly checked for their
germination, usually up to seven days. Data generated is used to calculate
percentage germination, which is often used for validating the existence of
allelopathy in natural or in agro-ecosystems (Anjum and Bajwa, 2005). Rather, proof
of allelopathy as a natural phenomenon requires a far more complex approach which
should consider the production and exudation of allelochemicals by the donor
species, the fate of the compounds in the environment in which they are released, as
well as the uptake and growth responses of the acceptor species (Leather and
Einhellig, 1988). Most studies on allelopathy in particular those based on bioassays,
only achieve ―proof of concept‖ by providing evidence that plants exhibit allelopathic
potential.
1.2.5.1b Plant growth bioassays
Bioassays that assess plant growth for a significant period of a plant‘s life cycle are
not used as often as bioassays that run for short periods (days rather than weeks),
but they all aid in contributing to understanding of the overall effect allelochemicals
have on plant growth (Hoagland and Williams, 2003). Preparation or collection of
foliar and root exudates (leachate), followed by growth bioassays and quantification
of allelochemicals in the various media, are commonly used techniques to study the
release of allelochemicals by donor species and their biological effects on acceptor
species. In addition, allelochemicals should be collected with the least disruption of
the normal mode of release. In some cases plants are grown hydroponically in water
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or in a porous medium (sand or soil), which is amended with allelochemicals or
extracts. Since the plants are not grown in sterile conditions, metabolism of the
compounds or conversion of compounds to a nonactive or even a more active state
is always a possibility (Inderjit and Nilsen, 2003).
1.3 Conyza species
Conyza bonariensis, Conyza sumatrensis and Conyza canadensis commonly known
as flax-leaf fleabane, tall fleabane and Canadian fleabane respectively, belong to the
sunflower (Asteraceae) family. About 7% of the species listed as declared invaders
and weeds in South Africa belong to the Asteraceae family (Henderson, 2001).C.
bonariensis, C. sumatrensis and C. canadensis are closely related species, and
therefore they do not differ much in their morphology during early growth stages.
Characteristics that set the three species apart are mainly detail related to leaf
morphology of mature plants, the flowers and seed.
1.3.1 Botanical description
1.3.1.1 Taxonomy
Division
Magnoliophyta (Flowering Plants)
Class
Magnoliopsida (Dicotyledons)
Subclass
Asteridae
Order
Asterales
Family
Asteraceae (Aster family)
Genus
Conyza Less (horseweed)
Species
Conyza bonariensis (L.)Conquist
(Flax-Leaf fleabane)
Conyza sumatrensis (L.)Conquist
(Tall fleabane)
Conyza canadensis (L.)Conquist
(Canadian fleabane)
1.3.1.2 Biology and ecology
C. sumatrensis (Figure 1.1) is native to South America, and is a nearly unbranched
semi-woody, annual plant that grows to more than 2 m in height, with a sturdy
taproot, and stems with short, dense green hairs (Botha, 2001). Side branches only
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occur on the upper third of the plant and are never longer than the main stem. The
leaves are simple, usually alternate, with pointed tips, and narrow gradually to the
base. Leaves are approximately 10 cm long and 1.5 cm wide. Flowers are white, in
heads of up to 4mm in diameter that occur in large, terminal plumes. The fruits are
straw-coloured, flattened and short-haired. The pappus consists of persistent hairs
and is up to 4 mm long (Ivens et al., 1978; Botha, 2001).
Figure 1.1 Conyza sumatrensis (from: Weeds of Crops and Gardens in Southern
Africa, Ciba-Geigy (1985))
Also originally from South America, C. bonariensis (Figure1.2) is an erect,
multibranched, semi-woody plant that grows up to 1.2 m high, with a taproot system
(Botha, 2001). The stems are short-haired, greenish and finely corrugated. The side
branches are always longer than the main stem. Leaves are narrow, crinkled,
greyish in colour, slightly toothed around the edges, up to 10 cm long and 1cm wide.
The flowers have small yellowish heads at the ends of the branched stem at the
upper part of the plant. Fruits are straw-coloured, up to 1.5 mm long, flattened and
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sparsely haired. The pappus consists of persistent hairs and is up to 5 mm long
(Botha, 2001; Shrestha and Hembree, 2005).
Figure 1.2 Conyza bonariensis (from: Weeds of Crops and Gardens in Southern
Africa, Ciba-Geigy (1985))
C. canadensis (Figure 1.3) believed to be of North American origin, is an erect, semiwoody, winter or summer annual plant, with a short taproot. The stems are 1.8 m
high, nearly smooth or bristly hairy, unbranched at the base, branched near the top,
with many small flower heads. Stem leaves are alternate, numerous and crowded on
the stem, often appearing opposite or even whorled, lanceolate to linear, with nearly
entire margins; upper stem leaves only 5 mm wide (Weaver, 2001). Pale green to
yellowish colour is given as a characteristic of this species. Flowers are 5 mm in
diameter with white or slightly pink ray flowers and yellow disk flowers (Frankton and
Mulligan, 1987; Alex, 1992). The fruits are straw-coloured, flattened and short-
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haired. The pappus consists of persistent hairs and is up to2-4 mm long (Holm et al.,
1997).
Figure 1.3 Conyza canadensis (from: Weeds of Crops and Gardens in Southern
Africa, Ciba-Geigy (1985))
Conyza spp are prolific seed producers. C bonariensis reportedly produces about
375561 seeds per plant (Kempen and Graf, 1981). Seeds are dispersed within one
or two capitula, for which time to maturity depends on the climate (Thebaud et al.,
1996). Primary dispersal of Conyza seeds is via wind. There is no long seed
dormancy in Conyza, with viability estimated as 1 to 2 years in the field (Weaver,
2001).
The optimum temperature regime for germination is 10°C minimum and
25°Cmaximum (Zinzolker et al., 1985). Conyza spp are small-seeded. Seeds only
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emerge from (or near) the soil surface. For this reason the occurrence of Conyza is
more common in zero or reduced till systems where the majority of seed remain on
or close to the soil surface, and where increased stubble cover keeps the soil
surface wet for longer (Wu and Walker, 2004). Although very limited emergence
occurs in mid-winter, young autumn or early winter seedlings actively grow during
winter despite cold and dry conditions (Weaver, 2001). Even where there does not
seem to be much growth aboveground, root growth progresses. The building of a
root system during winter provides sufficient food reserves for rapid growth during
the following spring.
Due to difficulty in identification and confusion of these three species of Conyza,
particularly because they grow in mixed populations allowing the occurrence of
intermediate forms, hybridization has been speculated by some researchers
(Thebaud and Abbott, 1995; Anzalone, 1964; Melzer, 1996). The differences of the
three species have been explained as follows (Milovic, 2004):
C. canadensis differs from C. bonariensis and C. sumatrensis by having a
shorter, nearly glabrous involucre (3-4 mm long), only 25-40 female florets per
capitula and having marginal florets with short (up to 1 mm) but well
developed ligula.
C. sumatrensis differs from closely related species C. bonariensis mostly by
the marginal florets in the capitula. In the species C. sumatrensis marginal
female florets are zygomorphic, while in C. bonariensis all the florets are
actinomorphic. C. sumatrensis is otherwise much taller, branched out only in
the upper part of the stem, lateral branches generally not overtopping the
main axis and the inflorescence is rhombic in outline (Weaver, 2001)
C. sumatrensis also has a greater number of leaves which are bigger, wider
and with ramified lateral veins (Pignatti, 1982; Poldini and Kaligari, 2000;
Sida, 2002). C. sumatrensis is recognisable particularly by its often welldeveloped winter rosettes (Anzalone, 1964, Poldini and Kaligari, 2000).
The genus Conyza represents one of the foremost examples of intercontinental plant
invasions from the New World to the Old World. C. sumatrensis and another species
of the genus are considered the most widespread species throughout the world
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(Thebaud and Abbott, 1995). According to Hao et al. (2009), its invasiveness was
always underestimated because of the difficulty in distinguishing the species from
Conyza bonariensis in the field.
Widderick and Wu (2009) suggested the following as factors that make Conyza
species major weeds.
Conyza spp are major weeds of fallow land. These species competes for soil
water and nutrients in both crop and fallow phases.
Conyza spp are difficult to control with herbicides. Inconsistent control is often
obtained with herbicide treatments, especially once plants in the rosette
growth stage exceed a diameter of 30 mm. Where fleabane becomes a
problem in fallows, weed control costs can increase by up to 80% due to the
difficulty of controlling it.
Conyza spp are capable of developing herbicide resistance.
C. bonariensis flower throughout the year.The pappus on the seed enables it
to be dispersed long distances by wind.
1.3.2 Distribution and habitat of Conyza spp in South Africa
In South Africa the first record of C. sumatrensis (formerly known as Conyza albida)
was in 1896 from the Cape Peninsula. Other records of the plant include: White
River district 1965, Orange Free State 45 km SE of Kimberly 1969, Potchefstroom
1974, Transvaal Naboomspruit 1977, 10km North of Hazyview 1989, Cape Town
Ronderbosch 1989(Danin, 1990). These recordings indicate that the plant has
invaded most, if not all the provinces in the country. Records of this species in
neighbouring countries include Zimbabwe (1957), Namibia (1989), and Mozambique
(1958). C. sumatrensis is alleged to be more competitive than the other species of
Conyza (Thebaud et al., 1996). Case and Crawley (2000) stated that this species
prefers highly disturbed areas and has a capacity to establish in a native ecosystem.
In France, C. sumatrensis has been reported in gardens, vineyard, and in old fields
(Case and Crawley, 2000). While in the Mediterranean, it typically grows and
persists in old fields whose ages range from 20 to 30 years of abandonment
(Thebaud et al., 1996). In England, C. sumatrensis has only been found in urban
habitats, such as in concrete paving, gravel car parks and building sites (Wuizell,
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1994). In West Africa it has been found near the edge of forests or in clearings as a
weed in perennial crops (Ivens et al., 1978).
According to De Wet (2005) the first report of the occurrence of C. bonariensis in
South Africa was made in May 1895 in Franschoek. Most recent reports of Conyza
species in the country have been on C. bonariensis, particularly in the Western
Cape. One such report is by Fourie and Raath (2009) who assessed the effect of
organic and integrated soil cultivation practices on the weed population in a vineyard
situated in the Paarl wine district. They found that of all the weeds present C.
bonariensis filled the niche in this crop the most effectively and became the dominant
species. In January 2003 a report of herbicide resistance in South Africa was made
when resistance occurred in C. bonariensis in the Breede Valley, South Africa
(Heap, 2005). The following year it was listed as one of the major weeds in South
Africa that are well established and have substantial impact on natural ecosystems.
In this survey it was also declared a riparian weed, which means that the number of
times it occurred in riparian ecosystem exceeds that of landscapes in this particular
survey (Nel et al., 2004). C. bonariensis has been documented as a host of insect
pests which attack crop plants in New Zealand and South Africa, where the weed
hosted mealiebug species, and in the Hex river valley where it was host to
Tetranychus urticae, which is a spider mite that attacks deciduous fruit, and causes
chlorotic spots (Fourie, 1996).
In a 1966 weeds survey on common weeds in South Africa conducted by Henderson
and Anderson, C. canadensis was reported to be distributed in the Transvaal,
Swaziland, Natal, Orange Free State, Basutholand, Northern Cape and Western
Cape. Information on the occurrence of C canadensis in South Africa in recent years
is scarce, although it has been reported to predominantly occur in the northern and
eastern parts of the Western Cape (De Wet, 2005). Globally, C. canadensis is a
weed of more than 40 crops (Holm et al., 1997). The list includes: fruit orchards,
vineyards, field crops such as maize, soybean and cotton, particularly where
conservation tillage or no-till systems are used, hay crops, pastures and rangeland
(Kapusta, 1979; Buhler, 1992; Wiese et al., 1995;Leroux et al. 1996).C. canadensis
has also been implicated in serving as a host to insect pests, such as the tarnished
plant bug (Lygus lineolaris) and the alfalfa plant bug (Adelphocoris lineolatus).
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1.3.3 Interference and allelopathic potential of Conyza species
Allelochemicals released from plants often play a vital role in influencing the
vegetational composition and population structure of a site (Shaukat et al.,
2003).Intraspecific and interspecific competition of Conyza species has been
explored. Thebaud et al. (1996) reported that the ability to absorb and utilise both
water and nutrient resources within a competitive environment was greater in
Conyza sumatrensis than in Conyza canadensis. Since this group of weeds has
often been seen to form dense, almost pure stands and can tolerate a variety of
habitats and environmental conditions (Economou et al., 2002), it is reasonable for
researchers to suspect allelopathy could be involved in the suppression of other
plants in the vicinity. However, very limited literature exists on investigations into the
allelopathic effects of leachates of different plant parts, as well as for compounds
isolated from Conyza species. Phytotoxic effects of aqueous extracts of C.
canadensis and C. sumatrensis have been observed on important crops such as
tomato (Lycopersicon esculentum), wheat (Triticum aestivum), maize (Zea mays),
millet (Pennisetum americanum), radish (Raphanus sativus), mungbean (Vigna
radiata) and oats (Avena sativa) (Economou et al., 2002; Shaukat et al., 2003;
Travalos et al., 2007).
1.3.4 Control measures
1.3.4.1 Mechanical control
For effective control of Conyza it is better to treat when small, at its early growth
stages when it is actively growing, but before stem elongation. Hand-pulling after
stem elongation is effective in light soils, but on heavier soils a hand-hoe is required
to prevent the plant breaking and regrowing from the base. Planting of perennials to
increase ground cover and the shading effect will help in reducing reinfestation (Wu,
2004). Soil tillage can completely control Conyza species without the use of
herbicides but the former practice is not always practical, especially in minimum- and
zero-tillage systems. Mowing has been reported to have the tendency to stimulate
additional branching from the crown and only delays seed production. It also hardens
the plants and makes control with post-emergence herbicides difficult.
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1.3.4.2 Biological control
In annual crop systems, biological control offers hardly any options for weed control
because of the requirement for absolute host-specificity in biocontrol agents, and the
weed spectrum on crop fields is mostly diverse. Very little information is available on
biocontrol options for Conyza species. However, the bacterium Pseudomonas
syringae pv tagetis has been reported to affect these weeds, but this potential
biocontrol agent has not yet been developed on a large scale (Charudattan, 2001).
1.3.4.3 Chemical control
The two most commonly used herbicides for the control of Conyza species are
paraquat and glyphosate (De Wet, 2005).However, many other herbicides were
listed in the 2012 Croplife (South Africa) Herbicide Module (Table 1.1).
Table 1.1 Herbicides registered to control Conyza species (Croplife South Africa,
2012)
Weed Species
Active ingredient
Recommended
Trade name
Crops
Trooper SL
Rate
Conyza bonariensis
280/80 gL-1
2,4-D/Dicamba
Grass
pastures;
Flax-leaf fleabane
Maize;
Sugarcane;
Kleinskraalhans
Wheat
Bromoxynil/Ioxynil
200/200 gL-1
Voloxytril 400 EC
Sugarcane
Carfentrazone-Ethyl
400 gKg-1
Aurora 40 WG
Almonds;
Aloes;
Avocadoes;
Barley;
Lawns;
Turf;
Apples;
Bananas;
Citrus;
Coffee;
Granadilla; Grapes; Guavas;
Hops;
Kiwi;
Litchi;
Macadamias;
Mangoes;
Nectarines; Olives; Papaya;
Papaya; Peaches; Pears;
Pecans; Plums & Prunes;
Tea; Wheat
Dicamba
Diuron
700 gKg
-1
Dominator
Grain sorghum; Wheat
800 gKg
-1
Karmex
Citrus;
Pineapples;
Sugarcane
300/100 gL-1
Diuron/Paraquat
Volmuron
Bananas; Citrus; Papaya;
Sugarcane
Conyza canadensis
Canadian fleabane
Acetochlor/Ametryn
Ametryn
-1
450/250 gL
-1
500 gL
Acetamet 700 SC
Sugarcane
Ametryn 500 SC
Bananas;
Kanadese skraalhans
Pineapples;
Sugarcane
Glufosinate-
200 gL-1
Basta
Ammonium
Almonds;
Avocadoes;
Aloes;
Apples;
Bananas;
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Barley;
Citrus;
Coffee;
Granadilla; Grapes; Guavas;
Hops;
Kiwi;
Litchi;
Macadamias;
Mangoes;
Nectarines; Olives; Papaya;
Papaya; Peaches; Pears;
Pecans; Plums & Prunes
Glyphosate
500 gKg
-1
-1
Metribuzin
480 gL
Kilo WSG
Afforestation; Firebreaks
Metribuzin 480
Asparagus;
Lucerne;
Leguminous
pastures;
Potatoes;
Sugarcane;
Tomatoes
500 gL-1
Simazine
Simazine
Apples; Asparagus; Canola;
Citrus; Grapes; Pears
-1
Tebuthiuron
Conyza sumatrensis
50 gKg
-1
2,4-D/Dicamba
280/80 gL
Spike 50 GR
Sisal
Trooper SL
Grass
pastures;
Tall fleabane
Maize;
Sugarcane;
Vaalskraalhans
Wheat
Atrazine/Sulcotrione
300/125 gL-1
Lawns;
Turf;
Caravelle
Maize; Sweetcorn
500 gKg
-1
Kalash 700 WSG
Most Agricultural Situations
Hexazinone
750 gKg
-1
Velpar DF
Afforestation; Sugarcane
Simazine
500 gL-1
Simazol SC
Apples; Asparagus; Canola;
Tebuthiuron
50 gKg-1
Spike 50 GR
Glyphosate
Citrus; Grapes; Pears
Sisal
1.3.4.4 Herbicide resistance
Herbicide resistance can be defined as the inherent ability of a weed to survive a
rate of herbicide which would normally result in effective control (WSSA, 1998).Most
cases of herbicide resistance have occurred in situations where the same herbicides
(or herbicides with the same mode of action) have been used repeatedly over a
period of years(De Wet, 2005). Herbicide resistance can result from any inherited
trait, which allows plant to survive herbicide applications. This could be due to
biochemical or physiological changes, morphological alterations that affect herbicide
uptake or interception or phonological changes, such as changes in germination
patterns. Herbicide resistance is generally thought to occur within weed populations
as a consequence of the intense selective pressure exerted by lack of diversity in
weed management practices (Gressel and Segel, 1978).
In South Africa herbicide resistance was reported for the first time two decades ago
in the Western Cape, when Cairns and Laubscher (1986) reported resistance of wild
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oats (Avena fatua) to diclofop-methyl (De Wet, 2005). Pieterse (2010) states that
following this report, other reports of herbicide resistance in grass species increased
dramatically. Botes and Van Biljon (1993) showed multiple resistance of ryegrass (L.
rigidum) to ACCase and ALS inhibitors (Heap, 2009). These findings were confirmed
five years later (Smit and De Villiers, 1998; Smitet al., 1999).The occurrence of
resistance in smooth pigweed (Amaranthus hybridus) to triazine in 1993 reported by
Botes and Van Biljon was the first record of herbicide resistance in broadleaved
weeds in South Africa (Heap, 2009; Pieterse, 2010). In recent years wild radish has
shown indications of resistance to chlorsulfuron, and several other ALS inhibitors
(Smit and Cairns, 2001; Heap, 2009; Pieterse, 2010).
Herbicide resistance has evolved within Conyza populations in several countries
(VanGessel, 2001). Weed resistance to paraquat and glyphosate have been
reported in Conyza bonariensis and Conyza canadensis. Paraquat is a foliage-active
bipyridylium herbicide, which exerts its phytotoxic effect by catalyzing electron
transfer from PSI to molecular oxygen-generating superoxide anion radicals and
other active oxygen species. These phytotoxic oxygen species cause lipid
peroxidation
and
membrane
damage
(Raczet
al.,
2000).
Glyphosate
[N-
(phosphonomethyl) glycine] is a non-selective, broad spectrum, systemic, postemergence herbicide. This herbicide kills weeds by metabolic disruptions in the plant
(Franz et al., 1997). It inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate
synthase (EPSPS) which is essential for biosynthesis of certain aromatic amino
acids (Mueller et al., 2003).
A paraquat resistant biotype of Conyza originated in the Tahrir irrigation area in
Egypt (Shaaltieland and Gressel, 1986). An intensive paraquat spraying program
was undertaken in vine and citrus plantations in 1970 and difficulties in controlling
this weed were first observed in the mid-1970s (Fuerst et al., 1985). The exact site
and mechanism of paraquat binding to sequester the herbicide remains to be
determined, but Fuerst et al. (1985) proposed that it is primarily due to exclusion of
the herbicide from the site ofaction in the chloroplast, resulting from rapid
sequestration viaan unknown mechanism.
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The first reported cases of glyphosate-resistant C. bonariensis were in South African
orchards and vineyards in January 2003(Figure 1.4).Reports were received from the
Breede Valley (about 100km north east of Cape Town) of glyphosate failing to
control C. bonariensis at registered dosage rates (Heap, 2005; Heap, 2009).Other
cases that followed were in 2004 and 2005 in Spanish and Brazilian orchards (Heap,
2007). The glyphosate resistance mechanism in C. bonariensis is still unknown to
date, as no literature is available on this topic (Dinelli et al., 2008). According to
Pieterse (2010), a biotype of C. canadensis from the Limpopo province that was
resistant to paraquat was also recorded (Dr PJ Pieterse,Department of Agronomy,
University of Stellenbosch: unpublished results).
Figure 1.4 A map showing the distribution of glyphosate and paraquat resistant C.
bonariensis in the Western Cape, South Africa (Prof. A Cairns, unpublished; De Wet,
2005)
A biotype of C. sumatrensis resistant to imazapyr was discovered on a farm in the
province of Seville, Spain, on land that had been continuously treated with this
herbicide (Osuna and De Prado, 2003). Imazapyr is a non-selective herbicide
belonging to the imidazolinone family, used for the control of a broad range of weeds
including annual and perennial grasses and broad-leaved species. The mode of
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© University of Pretoria
action of imazapyr is the inhibition of acetoacetate synthase, the first common
enzyme in the biosynthesis of the branched-chain amino acids valine, leucine and
isoleucine (Saari and Mauvais, 1996).
According to anecdotal evidence, there is currently uncertainty about which Conyza
spp occur where in South Africa. A certain school of thought believes that C.
canadensis might not occur in the Western Cape. As mentioned above Pieterse
tested a C. canadensis from Limpopo Province, which is about 2000 km removed
from the Western Cape. This begs the question, what is the real situation with
regards the distribution of Conyza spp in South Africa.
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CHAPTER 2
ALLELOPATHIC INFLUENCE OF CONYZA BONARIENSIS ON LETTUCE AND
TOMATO SEED GERMINATION AND EARLY SEEDLING DEVELOPMENT
2.1 Introduction
Conyza spp are annual, herbaceous, invasive weeds of the Asteraceae family.
Worldwide, three Conyza spp (Conyza bonariensis, Conyza canadensis, and
Conyza sumatrensis) are noxious weeds in many crops (Everett, 1990; Weaver,
2001; Milovic, 2004). In South Africa, these three species are well known weeds and
were first noticed about a century ago, with infestations of one or more species in
every province (Danin, 1990; Botha, 2001). C. bonariensis is a weed of cultivated
and non-cultivated lands, gardens, roadsides and waste places (Ciba-Geigy, 1985;
Botha, 2001). Adding to its problem status is the recent discovery that certain C.
bonariensis biotypes in South Africa and other parts of the world are resistant to the
herbicide glyphosate, and in certain cases to both glyphosate and paraquat
(Pieterse, 2010). Despite its invasiveness and ability to compete severely with crops,
little is known about the mechanisms of interference employed by C. bonariensis.
The phenomenon of allelopathy is known to be one of two predominant forces in the
development of plant communities and spatial patterns therein (Rice, 1984). Among
the weed species reported globally, a considerable number are known to possess
allelopathic potential (Ashraf and Sen, 1978; Shaukat et al., 1983; Ahmed and
Wardle, 1994). To date, very few studies have assessed the allelopathic potential of
C. bonariensis. However, studies on a related species C. canadensis, identified three
active
enyne
derivatives,
(2Z,8Z)-matricaria
acid
methyl
ester,
(4Z,8Z)-
matricarialactone, and (4Z)-lachnophyllum lactone (Queiroz et al., 2012). According
to Queiroz et al. (2012), the three isolated acetylenes may be involved in the
reported allelopathy of C. canadensis, and that since these compounds are found in
other Asteraceae plants, they may play a role in allelopathic properties of different
species in this family. In their study, it was proposed that (4Z)-lachnophyllum lactone
showed most promise as a potential herbicide. In preliminary studies investigating
the allelochemical characteristics of C. sumatrensis another closely related species
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of C. bonariensis, it was found to have an inhibitory effect on oat germination and
seedling growth (Shaukat et al., 1983; Economou et al., 2002).
Allelopathy can be seen as a problem, or if viewed positively, can serve as a weed
management tool for sustainable agriculture (Weston and Duke, 2003; Ferreira and
Reinhardt, 2010; Bezuidenhout et al., 2012). Because the potential of undesirable
environmental contamination from herbicides is high in some instances, researchers
have suggested the use of plant-produced secondary metabolites as natural
pesticides in agriculture or as structural leads for new synthetic pesticides, which are
environmentally safe and are equivalent in terms of efficacy and selectivity to the
currently available synthetic herbicides (Putnam et al., 1983; Travalos et al., 2007;
Queiroz et al., 2012).
When considering the allelopathic potential of plants, it is imperative to distinguish
between the effects of competitive and chemical (allelopathy) interference (Fuerst
and Putnam, 1983; Leather and Einhellig, 1986; Inderjit and Olofsdotter, 1998).
Therefore, bioassays in allelopathy research should be designed to eliminate the
effects of plant-plant competition. Laboratory bioassays allow researchers to
eliminate possible alternative interferences through controlled experimental designs
and manipulation of nearly all parameters, in order that investigators can vary
complex field conditions one at a time in the search for mechanistic interactions.
C. bonariensis plants in South Africa are often observed to form dense, almost pure
stands; therefore, it is conceivable that this species could employ both competition
and allelopathy in the suppression of other plants in the surrounding area. Because
little is known about the allelopathic nature, as compared to competition effects of
this weed, experiments in the present study were designed to assess the allelopathic
potential of C. bonariensis. The objectives of the investigation were to: (1) evaluate
the effect of aqueous extracts of C. bonariensis on seed germination and seedling
growth of two test species; and (2)to establish if the compounds responsible for
germination and seedling growth response are polar or non-polar in nature by using
two solvents with differing polarities to extract C. bonariensis plant tissue.
Experiments in this bioassay approach were designed to minimise potential
interfering factors, e.g., competition, osmotic effects and pathogenic organisms.
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2.2 Materials and Methods
2.2.1 The bioassay technique
In general, extract bioassays are conducted in Petri dishes by placing seeds of
receiver or test species on substrata (often filter paper) moistened with aqueous
infusions or plant extracts of donor species (Wu et al., 1998). The Petri dishes, which
usually are placed in an incubator under controlled light and dark periods, are
regularly checked for seed germination, and early seedling development, often for at
least seven days. Data recorded are typically used to calculate percentage
germination and to determine early seedling growth. Results are employed to make
inferences on allelopathy in natural ecosystems or in agro-ecosystems.
2.2.1.1 C. bonariensis material used in the study
Mature plants of C. bonariensis were collected on the Hatfield Experimental Farm of
the University of Pretoria. Test species were tomato and lettuce. According to
Reinhardt et al. (1999), the type, amount and location of allelochemicals may play an
important role in the determination of a plant‘s allelopathic potential. Leaf and root
material of C.bonariensis collected at the pre-flowering stage were used in all
experiments. The highest content of inhibitors (allelochemicals) is reportedly usually
present in the leaves of a plant (Roshchina and Roshchina, 1993). It has also been
observed that phytotoxic activity of upper leaves and inflorescence of related species
C. sumatrensis is significantly higher than in other tissues studied, e.g., in stems
(Economou et al., 2002). Therefore, it was assumed that the leaf material used in
these studies was probably the richest in potential inhibitors.
2.2.1.2 Preparation of crude extracts
After sampling, the plant material was frozen immediately and then freeze-dried prior
to extraction. Allelopathic bioassays with ground and frozen plant material have
received a great deal of criticism, for the reason that grinding results in the release of
certain compounds, which may not be released under natural circumstances. It is
possible that the extraction procedure may cause qualitative and quantitative
changes in the phytochemical profile of the plant material. We considered the freezedrying process and subsequent non-drastic extraction as practical for demonstration
of allelopathic potential.
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For germination bioassay, leaf and root extracts were prepared by extracting 50 g of
C. bonariensis leaves or roots with 350 ml of two solvents: pure water (polar) and
hexane (non-polar). Plant material was extracted separately, and not consecutively,
with the two solvents. Plant-solvent mixtures were stirred, covered with aluminium
foil and placed in the dark for 24 h at room temperature. Extract solutions were
filtered through Whatman No.1 filter paper and diluted with the respective solvents to
give a concentration range of 25, 50, 75, and 100% (v/v). The control treatment was
distilled water. Aliquots of 5 ml of each of the extract solutions were added to filter
paper in Petri dishes. For the hexane treatments the solvent was allowed to
evaporate off the filter paper before 5 ml distilled water was added to the Petri
dishes, each containing 10 seeds of either lettuce or tomato that had been sterilized
beforehand in 1% sodium hypochlorite. Each treatment was replicated ten times.
Petri dishes were sealed with Para-film-® and stored in a growth chamber at 25ºC
(12h/12h light/dark) for seven days. Seed germination was recorded every day, and
root (radicle) and shoot length were measured on day 7 only.
2.2.1.3 Choice of test species
Lettuce and tomato were the chosen test species. Many similar bioassay studies
have used lettuce and tomato as test species because of their known germination
and growth behaviours. Most often lettuce (Lactuca sativa L.) is used to simulate
plant response to allelochemicals because of its fast germination and high sensitivity
(Rasmussen and Einhellig, 1979; Leather, 1983; Yu and Matsui, 1994; Macias et al.,
2000). It is used extensively in allelopathy studies and allows comparison of
bioassay results for many different compounds.
2.2.1.4 Germination and seedling development assessments
Seeds of all acceptor species were treated alike. Germination of all seeds was
determined at set times in order that no discrimination could be made between
acceptor species as to length of time needed to germinate and to develop. Seeds
were considered to have germinated if their radicles had emerged and were at least
1 mm in length. Seeds were tested prior to being bioassayed for viability to ensure
optimum germination rates.
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2.2.1.5 Sterilisation procedures
To ensure the exclusion of microbial contamination that have the potential to cloud
the results the following measures were introduced:
Distilled water was autoclavedat121 ºC for 30 minutes.
Sterilised Petri dishes and filter paper from sealed boxes were used in the
experiments.
All experiments were conducted in a laminar flow cabinet, where aseptic
conditions were maintained by swabbing surfaces and instruments with 70%
ethanol and through flaming.
Fungicide-coated seeds of lettuce variety Great Lakes and tomato variety
Moneymaker were surface-disinfected by soaking them in 1% commercial
bleach for twenty minutes. The seeds were then rinsed three times with
sterilized distilled water and air-dried under laminar flow.
2.2.2 Exclusion of osmotic potential effects on germination and growth
It is often assumed that the response of seed or seedlings to plant extracts is due
entirely to allelopathy, however, the possibility exists that the extracts may also exert
negative osmosis effects on the test species (Bell, 1974), and some investigators
have assessed the relative importance of osmotic influence and allelopathic potential
of plant extracts on seed germination and early seedling development (Stowe, 1979;
Bothma, 2002; Dixon, 2008). Osmotic effects are well known to induce stress
responses in plants, primarily by causing dehydration of plant material (Slayter,
1967).In the case of seed exposed to water that contain dissolved materials, high
osmotic potential (low water potential) could limit or prevent water imbibition by the
seeds required for germination.
2.2.2.1 Measuring principle in determining osmotic potential
In this study the Herman Roebling digital micro-osmometer was used. The principle
of its operation is that freezing point depression below that of pure water is a direct
measure of the osmotic concentration of an aqueous solution. Pure water freezes at
0°C, whereas an aqueous solution with an osmolality of 1 Osmolkg-1water freezes at
−1,858°C. The sample starts off at room temperature. It is pipetted into a sample
tube, which is placed onto the measuring head. The measuring head is pushed
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beneath its guide rod, thus inserting the sample tube into the cone shaped cooling
aperture. Now the sample begins to be cooled. The digital display will show
decreasing values. Once zero is reached, increasing negative values will be
displayed. At a certain stage of supercooling (when the digital display reads −70°C),
a cooled needle is inserted manually to initiate ice formation. The temperature will
begin to rise until the freezing point is reached. The point of disparity thus achieved
is the value. The digital display of the machine will display milliOsmol and not °C,
because osmolality is directly related to freezing point reduction.
2.2.2.2 Use of polyethylene glycol in studying osmotic potential effects
As mentioned above, the aim of this experiment was partly to demonstrate that
osmotic effects could cloud allelopathic effects in bioassays employing seed
germination and early seedling development as parameters for allelopathic effects.
The same procedure was not followed in the case of tomato test species because
the aim was to demonstrate the principle that cognisance ought to be taken of
osmotic potential in bioassays of this nature, and for this purpose lettuce served as
test case.
Polyethylene glycol (PEG−6000) is commonly used for testing plant responses to
osmolalities of substrates. It affects seed germination only by altering the osmolality
of water such that any effect observed on the germinating seed is a result of osmotic
potential of the solution. An osmotic range was prepared by dissolving different
amounts of PEG−6000 in distilled water. It has been previously determined that
concentrations of 12.5, 25, 50, and 75 gL-1water of PEG would give the best osmotic
range for bioassay studies (Hoagland and Brandsaeter, 1996). However, in this
study 100 and 125 g of PEG-6000 were included because osmolality recorded for
the aqueous weed extracts exceeded that provided by PEG-6000 concentrations
ranging from 12.5 to 75 gL-1.
Therefore, in order to exclude negative osmosis as a possible cause of lettuce seed
germination inhibition, osmolalities of C. bonariensis aqueous extracts were
measured in a preliminary experiment using the Herman Roebling digital microosmometer. This was done only in the case of the lettuce aqueous extracts since the
hexane extracts are not water soluble.
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2.2.3 Statistical analysis
Analysis of variance (ANOVA) was done using the statistical program SAS 9.2
(2002). A completely randomised design was used in all experiments. Analysis of
variance was used to test for differences between treatments. Radicle length data
for lettuce exposed to extract solutions were subjected to rank transformation,
otherwise the shoot and radicle data were acceptably normal with homogenous
treatment variances. In the case of germination percentages, angular transformation
was used to stabilise variances. Treatment means were separated using Tukey‘s
studentised range test least significant difference (LSD) at the 5% level of
significance.
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2.3 Results and Discussion
2.3.1 Effect of osmotic potential on lettuce seed germination and early seedling
development
When considering the influence of osmotic potential of PEG-6000 solutions on
lettuce germination, experiments showed that no significant inhibition of germination
occurred at any of the osmolalities created with PEG-6000 (Table. 2.1). Osmotic
potential did not interfere with radicle growth of lettuce up to and including 50
mOsmkg-1, the second highest osmolality tested. However, shoot length seems to
have been more sensitive, as significant inhibition occurred at the lowest osmolality
tested (24 mOsm kg-1).
Table 2.1 The effect of PEG-6000 solutions of increasing osmolality on germination
and radicle and shoot lengths of lettuce seedlings
PEG-6000
concentration gL
Osmolality
-1
-1
Percentage
Radicle length Shoot length
(mOsm kg )
germination (mm)
(mm)
0
0
100a
53.7a
18.5a
50
24
98a
51.5a
14.5b
75
50
97a
50.1ab
7.1c
100
96
98a
43.2b
6.9c
125
147
96a
34.4c
5.6c
Means in each column followed by different letters are significantly different
according to Tukey‘s Studentised Range test LSD (P< 0.05).
Based on the above findings, the significant reduction in germination of lettuce seed
that occurred as a result of exposure to C.bonariensis leaf aqueous extracts, was
attributable to a possible allelopathic effect and not to osmotic potential effects
(Table 2.2). Radicle length was significantly inhibited by an infusion concentration of
72 mOsm kg-1, which probably is due to possible allelopathic effects and osmotic
effects, whereas the significant reduction in shoot length that occurred from 110
mOsm kg-1 is probably due to a combination of allelopathic and osmotic effects.
Although germination was not significantly affected by osmotic effects, radicle and
shoot growth were at least partly influenced by osmotic potential.
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Table 2.2 Effect of C. bonariensis leaf infusions of increasing osmolality on
germination and mean radicle and length of lettuce seedlings
Extract
Osmolality
Percentage
Radicle
Shoot
concentration
(mOsm kg-1)
germination
length (mm)
length (mm)
0
0
97a
24.5a
13.8a
25
36
97a
20.8a
11.8ab
50
72
86a
12.5b
8.3ab
75
110
62b
6.0c
3.8c
100
138
56b
5.3c
5.5 bc
(%)
Means in each column followed by different letters are significantly different
according to Tukey‘s Studentised Range test LSD (P< 0.05).
Based on the data in (Table 2.1) increasing osmolality of PEG-6000 did not affect
radicle growth adversely within the range of 24 to 96 mOsm kg-1. Therefore, it is
possible that at the highest two osmolality (96 and 147mOsm kg-1), osmotic effects
may have interfered with radicle growth. As the osmolalities of the aqueous
infusions, prepared from C. bonariensis leaf material (Table 2.2), up to 36mOsm kg-1
were below the limit for growth inhibition in the PEG-6000 experiments, it can be
concluded that, apart from the three osmolalities (72,110 and 138mOsm kg-1)
osmotic effects did not play a role in the inhibitory effects of C.bonariensis infusions
on seed germination and seedling growth. Considering that growth inhibition of
lettuce shoots occurred at 24 mOsm kg-1 in the PEG-6000 experiments (Table 2.1), it
is highly probable that osmotic inhibition may have been a contributing factor from 36
mOsm kg-1 to 138 mOsm kg-1 in the shoot growth inhibition caused by C.bonariensis
aqueous leaf extracts, as illustrated in Table 2.2.
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2.3.2 Effect of aqueous extracts of Conyza bonariensis on germination and seedling
growth of two test species
2.3.2.1Effects of aqueous extracts on lettuce
Germination: Significant inhibition of seed germination was observed at the 75%
concentration for only the weed leaf infusion, and at 100% concentration for both the
leaf and root infusions. At 100% concentration the allelopathic effect of root extracts
was significantly higher than that of the leaves (Figure 2.1). As osmotic effects on
lettuce seed germination can be excluded, based on findings presented in Tables 2.1
and 2.2, germination inhibition observed here can be ascribed to possible
allelopathic effects.
Figure 2.1 Effect of aqueous leaf and root extracts of C. bonariensis on seed
germination of lettuce (Means with same letters do not differ significantly at P=0.05;
Comparisons were made across plant part and infusion concentrations; ANOVA
presented in Appendix A, Table A1)
Radicle growth: Figure 2.2shows that both leaf and root infusions of C. bonariensis
significantly inhibited radicle growth of lettuce from the 25% infusion concentrations
onwards. Overall, there is not a clear difference in the intensity of effects between
extracts of leaf and root material of C. bonariensis. It is important to note that the
osmotic potential results (Table 2.2) implicate that osmotic potential is playing a role
in plant responses from the 50% infusion concentration and higher.
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Figure 2.2 Effect of aqueous leaf and root extracts of C. bonariensis on root (radicle)
growth of lettuce (Means with same letters do not differ significantly at P=0.05;
Comparisons were made across plant part and infusion concentrations; ANOVA
presented in Appendix A, Table A2)
It needs to be emphasised that the perceived role of solution osmotic potential does
not mean that allelopathy had no role at 75 and 100% infusion concentrations
(Figure 2.2). The degree of inhibition tended to increase with increasing infusion
concentration, but was not significant in all instances. At 50% infusion concentration
only, the leaf infusion had a significantly greater effect on root growth reduction
compared to the control than the root infusion. Studies by Economou et al. (2002),
using similar methods of bioassay, showed an aqueous extract of dried aerial parts
of C.sumatrensis, another cosmopolitan species occurring in South Africa, to have
an inhibitory effect on the germination and seedling growth of oat (Avena sativa). Oat
radicle elongation was reduced with increasing extract concentration. Similar results
were reported from leachate experiments with Parthenium hysterophorus, a weed
also belonging to the Asteraceae family (Mersie and Singh, 1987). Therefore, results
from the bioassays using leaf extract of C. bonariensis agree with work done by
other researchers on Asteraceae species in relation to allelopathic potential.
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Figure 2.3Effect of aqueous leaf and root extracts of C. bonariensis on shoot growth
of lettuce (Means with same letters do not differ significantly at P=0.05; Comparisons
were made across plant part and infusion concentrations; ANOVA presented in
Appendix A, Table A3)
Shoot growth: In Figure 2.3 there is a trend for apparent growth stimulation between
25 and 75% infusion concentrations for both leaf and root extracts. At all the leaf
infusion concentrations, lettuce shoots were significantly longer than those of the
control and the 100% infusion concentration. This stimulatory effect was significant
for the root infusions at 25 and 50%. However, at 100% concentration the root
extract of the weed significantly reduced shoot growth of lettuce compared to the
control and all the other infusion concentrations. According to Belz et al. (2005)
some allelochemicals, which are toxic at high concentrations, can have a stimulatory
effect on one or several traits in a plant when applied at low concentrations. This
phenomenon is called hormesis.Belz et al. (2007) reported a significant hormesis
effect for Eragrostis curvula, with growth stimulation occurring at low parthenin
concentrations, and inhibition at higher doses. However it would take a far more
detailed experiment to prove this theory here. The significant inhibitory effect on
shoot growth of lettuce observed at 100% root infusion may at least partly be
attributed to osmotic potential considering the results presented in Table 2.2.
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2.3.2.2 Effects of aqueous extracts on tomato
Germination: Germination of tomato seed was significantly reduced by the leaf
aqueous extracts of C. bonariensis at 50% and higher infusion concentrations
(Figure 2.4). At 50 and 75% concentrations the leaf extract had a significantly greater
inhibitory effect than the root extract. At 100% infusion concentration tomato seed
exposed to the root extract did not germinate at all. A complete lack of germination
for tomato at 75% and 100% by shoot extracts of C. canadensis was also observed
by Shaukat et al. (2003).
Figure 2.4 Effect of aqueous leaf and root extracts of C. bonariensis on seed
germination of tomato (Means with same letters do not differ significantly at P=0.05;
Comparisons were made across plant part and infusion concentrations; ANOVA
presented in Appendix A, Table A4)
Radicle growth: The length of tomato radicles exposed to foliar and root extracts of
C.bonariensis were significantly reduced, and inhibition tended to increase with
increasing infusion concentration (Figure 2.5). At each of the 25%, 50% and 75%
concentrations, the leaf extract showed significantly greater inhibition than the root
extract. However, at 100% infusion concentrations there were no differences in
radicle length between the two extracts, largely as a result of very limited
germination. As in the present study, results showing that leaves contained the
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highest amount of phytotoxic substances were found by Picman and Picman (1984)
in P. hysterophorus.
Figure 2.5 Effect of aqueous leaf and root extracts of C. bonariensis on root growth
(radicle) of tomato (Means with same letters do not differ significantly at P=0.05;
Comparisons were made across plant part and infusion concentrations; ANOVA
presented in Appendix A, Table A5)
Shoot growth: For tomato shoots (Figure 2.6), there was a similar trend of growth
stimulation as in lettuce shoots between 25% and 50% infusion concentrations of the
leaf extract. However, this stimulation was only statistically significant at 25%.As
stated previously; this effect could be due to hormesis, which was not investigated
further in this study. At 75% infusion concentration, the leaf extract completely
inhibited shoot growth of tomato and the root extract significantly reduced growth
compared to the control. The 100% infusions of both plant extracts completely
inhibited shoot growth of tomato.
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Figure 2.6Effect of aqueous leaf and root extracts of C. bonariensis on shoot growth
of tomato (Means with same letters do not differ significantly at P=0.05; Comparisons
were made across plant part and infusion concentrations; ANOVA presented in
Appendix A, Table A6)
2.3.3 Effects of hexane extracts of leaves and roots of C. bonariensis on germination
and early seedling growth of two test species
2.3.3.1Effects of hexane leaf and root extracts on lettuce
Germination: Inhibition of lettuce seed germination by leaf and root hexane extracts
of C. bonariensis occurred only at the 75 and 100% infusion concentrations (Figure
2.7). At 75% infusion concentration the roots inhibited germination relative to the
control, whilst at the 100% infusion concentration the observed inhibition effect was
as a result of the leaf extract.
Figure 2.7 Effect of hexane leaf and root extracts of C. bonariensis on root growth of
lettuce (Means with same letters do not differ significantly at P=0.05; Comparisons
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were made across plant part and infusion concentrations ANOVA presented
Appendix A, Table A7)
Radicle growth: The hexane extract of C. bonariensis leaves significantly reduced
lettuce radicle growth from the 25% infusion concentration onwards as compared to
the control, with the 100% infusion concentration causing the greatest inhibition
(Figure 2.8). The degree of inhibition was not always significant from one infusion
concentration to the next. Significant radicle reduction was only observed at 25%
and 75% infusion concentrations for the root extract. As in the aqueous extract
experiment, the leaves of C.bonariensis exhibited a higher phytotoxic effect than the
roots.
Figure 2.8 Effect of hexane leaf and root extracts of C. bonariensis on root (radicle)
growth of lettuce (Means with same letters do not differ significantly at P=0.05
Comparisons were made across plant part and infusion concentrations ANOVA
presented in Appendix A, Table A8)
Shoot growth: Unlike the aqueous extracts, the hexane extracts of C.bonariensis did
not stimulate the growth of lettuce shoots at any concentration (Figure 2.9). Shoot
growth of lettuce was significantly reduced by the hexane leaf extract of C.
bonariensis from50% infusion concentration onwards. The root extract at all
concentrations significantly reduced shoot growth relative to the control, with the
highest reduction at 100% infusion concentration.
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Figure 2.9 Effect of hexane leaf and root extracts of C. bonariensis on shoot growth
of lettuce (Means with same letters do not differ significantly at P=0.05; Comparisons
were made across plant part and infusion concentrations; ANOVA presented in
Appendix A, Table A9)
2.3.3.2 Effects of hexane leaf and root extracts on tomato
Germination: Inhibitory effects on tomato seed germination by hexane extracts of C.
bonariensis leaves only occurred at 100% concentration. However, germination was
significantly inhibited by hexane extracts of C. bonariensis roots from the 50%
concentration onwards when compared to the control (Figure 2.10). The greatest
inhibition of tomato seed germination occurred at 100% concentration of the root
extract.
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Figure 2.10 Effect of hexane leaf and root extracts of C. bonariensis on seed
germination of tomato (Means with same letters do not differ significantly at P=0.05;
Comparisons were made across plant part and infusion concentrations; ANOVA
presented in Appendix A, Table A10)
Radicle growth: Both the root and leaf extracts of C.bonariensis significantly inhibited
radicle growth of tomato from the 25% concentration onwards (Figure 2.11) when
compared to the control. At 75% concentration, the degree to which the leaf extract
inhibited shoot growth was significantly higher than that of the root extract.
Figure 2.11 Effect of hexane leaf and root extracts of C. bonariensis on root (radicle)
growth of tomato (Means with same letters do not differ significantly at P=0.05;
Comparisons were made across plant part and infusion concentrations; ANOVA
presented in Appendix A, Table A11)
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Shoot growth: The reduction of tomato shoot growth by C. bonariensis hexane leaf
extract was significant only at 100% infusion concentration (Figure 2.12). For the root
extract, compared to the control, significant inhibition was already observed at the
25% concentration.
Figure 2.12 Effect of hexane leaf and root extracts of C. bonariensis on shoot growth
of tomato (Means with same letters do not differ significantly at P=0.05; Comparisons
were made across plant part and infusion concentrations; ANOVA presented in
Appendix A, Table A12)
2.4 Conclusions
Results from the germination and initial seedling growth studies suggest that
C.bonariensis contains phytotoxic allelochemicals that can inhibit, or at least retard,
the germination and early seedling development of crop species. Based on the
evidence from the germination studies water extracts appeared to contain different
inhibitory substances (allelochemicals) to the hexane extracts, which in some cases
were inhibitory to germination and subsequent growth. Allelochemicals contained in
the leaves of the weed appear to be more potent than those in roots. The root
infusions may have had lower allelopathic potential than the leaves in this bioassay
experiment, but this could be different for roots excreting allelochemicals into the soil
under natural conditions, thus it should be considered that the contribution of
allelochemicals contained in roots may have been underestimated in the laboratory
bioassay. However, if the results from these experiments depict what happens in the
field, the practical consequence of inhibitory compounds present in the leaves is that
incorporation of C. bonariensis foliage into the crop seedbed may impede
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germination and early seedling development of the two crop species tested. This
means that the weed should not be allowed to attain significant biomass on crop
fields at any stage, irrespective of whether the crop is present or not. Competition for
growth resources is therefore not the only plant-to-plant interference mechanism
which C. bonariensis possesses, and hence, there is an additional imperative for
controlling this weed.
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CHAPTER 3
ASSESSMENT OF THE ALLELOPATHIC POTENTIAL OF CONYZA
BONARIENSISROOT EXUDATES
3.1 Introduction
Plant roots have several functions in plant growth and development, including:
anchorage, provision of nutrients and water, and production of exudates with growth
regulatory properties. For the purpose of this discussion, compounds with growth
regulatory properties will be referred to as allelochemical compounds. Root activity is
confined to the rhizosphere, where roots affect soil structure, aeration and biological
activity as they are the major source of organic inputs, and are also responsible for
depletion of large supplies of inorganic compounds (Bertin et al., 2003). Exudates of
roots are often released in large quantities into the soil rhizosphere from living root
hairs or fibrous root systems. Root-specific metabolites are released that have
critical ecological impacts on soil macro- and micro-biota, amongst other plants of
the same or different species. Through the exudation of a wide variety of
compounds, roots influence the soil microbial community in their immediate vicinity,
imparts resistance to pests, support beneficial symbioses, alter the chemical and
physical properties of the soil, and inhibit the growth of competing plant species
(Takahashi, 1984).
Root exudates represent one of the largest direct inputs of plant-produced chemicals
into the rhizosphere, and therefore, root exudates also likely represent the largest
source of allelochemical inputs into the soil environment (Bertin et al., 2003). Roots
also have the potential to influence the two mechanisms of interference, viz.
competition and allelopathy. For a number of plant species, root exudates play a
direct role in plant-plant interactions through phytotoxins (allelochemicals) involved in
mediating chemical interference, i.e., allelopathy.
Allelochemicals, the organic compounds involved in the phenomenon of allelopathy,
are likely released from live plants and residual plant matter in great chemical
diversity and at different concentrations into the environment by root exudation,
leaching from aboveground parts, volatilisation and/or by decomposition of plant
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material (Rice, 1984; Inderjit and Dakshini 1995; Gibson, 2002; Pisula and Meiners
2010). The ability of allelochemicals to persist in soil is determined by sorption to soil
colloids,
leaching and
chemical or microbial degradation (Inderjit,
1998).
Allelochemicals can be highly selective, in that they can influence the growth of only
one organism, or they can exhibit broad activity in influencing the growth of many
species. Their synthesis and exudation, along with increased overall root exudate
production, is typically enhanced by stress conditions that the plant encounters, such
as extreme temperature, drought and UV exposure (Pramanik et al., 2000; Inderjit
and Weston, 2003).
Various allelochemicals in root exudates can affect metabolite production,
photosynthesis, respiration, membrane transport, germination, root growth, shoot
growth, and cell mortality in susceptible plants (Weir et al., 2004). These effects on
plant physiology, growth, and survival may in turn influence plant and soil community
composition and dynamics. Allelopathic root exudates can mediate negative plantplant interactions only if present at sufficient concentrations to affect plant growth
and survival. Preparation of foliar leachate and root exudates followed by growth
bioassays and quantification of allelochemicals in the medium are commonly used
techniques to study release of allelochemicals in the growth medium of plants
(Inderjit and Callaway, 2003).
In the present study the following research questions were addressed: (a) do Conyza
bonariensis roots release chemicals with allelopathic potential that are capable of
influencing the growth of neighbouring plants?; and (b) does the inhibitory effect of
C. bonariensis root exudates depend on the concentration of the toxins exuded by
the roots and released into the growth medium? A hydroponic culture system was
used to investigate whether C. bonariensis possesses and releases, through its
roots, chemicals with allelopathic potential by growing it together with test species in
a nutrient solution, and using plant growth as measure of effect. To answer the
second question, an experiment was done to determine if leachate from C.
bonariensis affected the growth of test species exposed to different leachate
concentrations.
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3.2 Materials and Methods
3.2.1 Hydroponic experiments
3.2.1.1 C. bonariensis (Pretoria population)
The experiment was a completely randomized design. C. bonariensis plants were
collected at the rosette stage on the University of Pretoria‘s Hatfield experimental
farm, and were grown hydroponically together with lettuce seedlings (Figure 3.1).
This population will be referred to as the Hatfield population in the document. Lettuce
seedlings, in the two-leaf stage, were obtained from Die Tuinhoekie nursery, and
transplanted to the pots. Lettuce had been chosen previously in similar studies
because it is generally considered an allelochemical-sensitive species (Meyer et al.,
2007). The three treatments were: (i) one C. bonariensis plant placed in the middle
and two lettuce seedlings on either side; (ii) one lettuce plant per pot, and (iii) one C.
bonariensis plant per pot. Treatments (ii) and (iii) served as the crop and weed
control treatment. Treatments were repeated 10 times and in total there were 30
pots. Initially all pots contained 1100 ml of Hoagland‘s nutrient solution and every
second day the water lost via transpiration and evaporation was replaced with
nutrient solution to the level of the original volume. The nutrient solution in pots was
replaced every seven days in order to avoid discrepancies in nutrient supply
between treatments.
Figure 3.1 Hydroponic system used to study the effect of allelochemicals released
by the roots of C. bonariensis plants on lettuce seedlings
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Air was bubbled through the nutrient solution by an airline entering through a
separate hole in the styro-foam lid of the pots (Figure 3.1). This experiment was set
up in a glasshouse with natural light and temperature range of 18 to 28 ºC.
Electroconductivity and pH were measured every day to make sure no changes were
occurring in the nutrient solution.
When considering the allelopathic potential of plants, it is essential to distinguish
between the effects of competition and allelopathy (Fuerst and Putnam, 1983;
Leather and Einhellig, 1986; Inderjit and Olofsdotter, 1998). Thus, bioassays in
allelopathy research should be designed to eliminate the effects of competitive
interference from the experimental system. In the present study, the possibility that
effects on plant growth might be the result of interference by competition was
eliminated by supplying all the plants with the same amount of nutrient solution and
light, hence a hydroponic system was chosen. The trial ran for four weeks, and in the
fifth week all plants were harvested and fresh and dry mass of shoots (above-ground
parts) and roots were measured.
3.2.1.2C. bonariensis (two Western Cape populations)
This experiment was repeated with two biotypes of C. bonariensis collected in the
Western Cape at two different locations, namely: Naboomsrivier in the Breede River
valley, and Willow Creek Boerdery in Heatlievale (Figure 3.2). One of the two
populations was suspected to be resistant to the herbicide glyphosate, and the other
susceptible.
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Figure 3.2 Hydroponic system in which one C. bonariensis from either
Naboomsrivier or Willow Creek Boerdery were grown with two lettuce seedlings; C.
bonariensis and lettuce plants grown on their own served as control
Data from the two experiments were compared in order to establish differences in
the allelopathic effects of the three provenances of C. bonariensis. Although all three
provenances were identified as C. bonariensis, there were clear morphological
differences between plants of the Pretoria and Western Cape populations, in
particular with regard to leaf shape and size (Figures 3.1 and 3.2).
All data were subjected to analysis of variance (ANOVA) using the statistical
programme SAS 9.2 (2002), and mean separation was done with the least significant
difference test of Tukey at P=0.05.
3.2.2 Leachate experiment
The experiment was a completely randomized. C. bonariensis plants were
transplanted when at the rosette stage from a crop field on the Hatfield experimental
farm of the University of Pretoria, and grown to maturity in the glasshouse in pure
quartz sand medium at a density of one plant per pot. Two seedlings of either lettuce
or tomato were transplanted into separate pots also containing quartz sand (Figure
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3.3). For each test species there were 25 pots. Every morning 300 ml of nutrient
solution was added to the 15 pots with C. bonariensis plants, and the leachate
collected at the base of the pots (Figure 3.4). The collected leachate was combined
from all 25 pots immediately after watering and a dilution series of 0% (pure nutrient
solution serving as control), 25%, 50%, 75%, and 100% (undiluted leachate) was
prepared. Test plants were treated with 200 ml of each leachate concentration in the
series every second day. Harvesting of the trial was done four weeks after treatment
commenced and dry mass of shoots and roots were measured.
Data were subjected to analysis of variance and mean separation was done with the
least significant difference test of Tukey at P=0.05. Analysis of variance (ANOVA)
was done using the statistical programme SAS 9.2 (2002).
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Figure 3.3 Test species that were used in the C. bonariensis leachate experiment:
lettuce seedlings (left); tomato seedlings (right)
Figure 3.4 Leachate experiment for the assessment of allelopathic effects of
C.bonariensis; Mitscherlich pots with C. bonariensis plants (donor plants) were
supplied with pans at bottom for leachate collection
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3.3 Results and Discussion
3.3.1 Effect of root exudates released by C. bonariensis plants on growth of lettuce
plants in hydroponic system
3.3.1.1 Hatfield C. bonariensis population
Fresh mass: There were significant differences in shoot and root mass between
lettuce grown alone (control) and lettuce grown with C. bonariensis from Hatfield
(Figure 3.5). On average there was 83% growth reduction in the roots of lettuce by
the weed treatment, and 65% growth reduction in the case of lettuce shoots.
Figure 3.5 Shoot and root fresh mass of test species lettuce grown hydroponically
with C. bonariensis plants collected on the Hatfield experimental farm (Means with
same letters do not differ significantly at P=0.05; ANOVA presented in Appendix B,
Tables B1 and B2)
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Figure3.6 Root growth variation between the roots of lettuce grown alone (left) and
lettuce grown with C.bonariensis (right)
Figure 3.7 Root and shoot mass comparison between plants representing the
controls of C. bonariensis and lettuce (left side of ruler), and plants from the weedcrop combination treatment (right side of ruler)
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C. bonariensis plants grown with lettuce did not differ much from those grown alone;
except for the chlorosis in the leaves and the slight reduction in shoot mass (Figure
3.7). This is an indication that even though there was no drastic reduction in plant
mass of C. bonariensis plants, competition for nutrients probably took place and/or
lettuce had an allelopathic effect on the weed. In a study by Chon et al. (2005), to
determine lettuce allelopathic effects on seed germination and early seedling growth
of several plant species, results suggested that extracts or residues from lettuce
plants had potent allelopathic activity and that the activity differed depending on
cultivar, extract or fraction.
Dry mass: Shoot and root dry mass of lettuce grown with C.bonariensis from the
Hatfield experimental farm was significantly reduced compared to the control (Figure
3.8). There was a 61% and 85% reduction in leaf and root mass respectively.
Figure 3.8 Shoot and root dry mass of test species lettuce grown hydroponically with
C. bonariensis plants collected on the Hatfield experimental farm (Means with same
letters do not differ significantly at P=0.05; ANOVA presented in Appendix B, Tables
B3 and B4 )
This type of experiment has been previously been used to demonstrate potential
allelopathic effects. A study by Irons and Burnside (1982) revealed that sorghum
plants grown in nutrient solution in which sunflowers were previously grown were
significantly shorter and their fresh and dry mass less than for those grown in fresh
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nutrient solution. Jose and Gillespie (1998) investigated the effects of juglone (active
agent causing growth inhibition found in black walnut) on the growth and physiology
of hydroponically grown corn (Zea mays L.) and soybean (Glycine max L. Merr.).
They found that soybean was more sensitive to juglone than corn, and that root
relative growth was the most inhibited variable for both species, with reductions of
86.5 and 99% observed in corn and soybean, respectively. As far as we were able to
ascertain, the allelopathic potential of C. bonariensis or any closely related species,
have thus far not been demonstrated using hydroponic experiments.
3.3.1.2 Western Cape C. bonariensis populations
Fresh mass: Significant inhibition of lettuce shoot and root growth occurred when this
species was grown together with C. bonariensis sourced at two locations in the
Western Cape (Figure 3.9). The degree to which lettuce shoots and roots were
reduced by the two biotypes did not differ significantly. For lettuce grown with C.
bonariensis from Naboomsrivier there was a 71% and 64% reduction in shoot and
root mass, respectively. Lettuce grown with C. bonariensis from Willow Creek
Boerdery showed a 59% and 67% reduction in mass for shoots and roots,
respectively.
Figure 3.9 Shoot and root fresh mass of test species lettuce grown hydroponically
with two Western Cape provenances of C. bonariensis (Means with same letters do
not differ significantly at P=0.05; ANOVA presented in Appendix B, Tables B5 and
B6)
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Dry mass: Shoot and root dry mass of lettuce grown with C. bonariensis from the
Western Cape showed significant reductions compared to the control (Figure 3.10).
There were a 53% and 49% reduction in shoot and root growth, respectively, for
lettuce grown with C. bonariensis from Naboomsrivier. Lettuce grown with C.
bonariensis from Willow Creek Boerdery showed a 49% and 65% reduction in shoot
and root growth, respectively. Similarly, for fresh mass data, there were no
significant differences in the inhibitory effects of the two Western Cape populations.
Figure 3.10 Shoot and root dry mass of test species lettuce grown hydroponically
with two Western Cape provenances of C. bonariensis(Means with same letters do
not differ significantly at P=0.05; ANOVA presented in Appendix B, Tables B7 and
B8)
Although the findings reported in Chapter 2 pointed to leaves of C. bonariensis being
a more important source of allelochemicals than the roots, those results were
obtained at the earliest stages of test species development. Moreover, the donor
plants in Chapter 2 were not alive as was the case in this experiment, and
allelochemicals were obtained in an unnatural way, i.e., through either aqueous
infusion or extraction with an organic solvent. Therefore, it is conceivable that
different types of allelochemicals and concentrations were involved in the present
study that involved live donor plants that could actively exude allelochemicals into
the growth medium of acceptor species.
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3.3.2 Effect of C. bonariensis leachate on the growth of two test species
3.3.2.1 Effect of Pretoria C. bonariensis leachate on the growth of lettuce
Fresh mass: There was no significant growth reduction in lettuce shoots and roots
caused by C. bonariensis leachate at all concentrations tested (Figure 3.11). This
implies that the growth of lettuce was not affected by increasing leachate
concentrations.
Figure3.11 Shoot and root fresh mass of lettuce that was exposed to C.bonariensis
leachate concentrations ranging from 0 to 100% (Means with same letters do not
differ significantly at P=0.05; ANOVA presented in Appendix B, Tables B9 and B10
Dry mass: Dry mass of lettuce shoots and roots exposed to leachate collected from
C. bonariensis were not significantly reduced (Figure 3.12). Although there was a
trend for apparent growth stimulation for roots of lettuce at all leachate
concentrations relative to the control, these differences were not significant.
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Figure 3.12 Shoot and root dry mass of lettuce plants exposed C. bonariensis
leachate concentrations ranging from 0 to 100% (Means with same letters do not
differ significantly at P=0.05; ANOVA presented in Appendix B, Tables B11 and B12)
3.3.2.1 Effect of Pretoria C. bonariensis leachates on the growth of tomato
Fresh mass: As in the lettuce experiment, tomato plants treated with C. bonariensis
leachate showed no significant growth reduction in the fresh mass of shoots.
However, significant stimulation of root growth was apparent at 50 and 100%
leachate concentrations (Figure 3.13 and 3.14).
Figure 3.13 Shoot and root fresh mass of tomato plants exposed to different C.
bonariensis leachate concentrations ranging from 0 to 100% (Means with same
letters do not differ significantly at P=0.05; ANOVA presented in Appendix B, Tables
B13 and B4)
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Figure 3.14 A: Roots of tomato grown in pure nutrient solution; B: roots of plants
treated with 100% C. bonariensis leachate concentration
Dry mass: Dry mass data for tomato showed that significant stimulation of tomato
root growth occurred only at 100% leachate concentration when compared to the
control (Fig 3.14B and 3.15). For both fresh and dry mass the trend for apparent
stimulation of tomato shoots at 100% leachate concentration was not statistically
significant when compared to the control.
Fig 3.15 Shoot tops and root dry mass of tomato plants exposed to a range of C.
bonariensis leachate concentrations ranging from 0 to 100% (Means with same
letters do not differ significantly at P=0.05; ANOVA presented in Appendix B, Tables
B15 and B16)
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The inability of C. bonariensis leachate to reduce lettuce and tomato plant growth
could be due to two reasons; firstly, insufficient accumulation of putative allelopathic
compounds given the methodology and secondly, the duration of the experiment. To
elaborate on the first reason, it is important to note that the growth medium used in
this experiment was pure sand for both donor and test species. Although plants can
grow in pure sand, the latter does not have adsorptive capacity to bind water;
therefore, the retention of allelochemicals in the growth medium would likely also
have been negligible in this experiment, and hence, considerable allelochemical loss
through leaching likely took place. To support this notion of decreased concentration
of allelochemicals, for dry mass, apparent stimulation of tomato plant growth was
significant only at 100% leachate concentration instead of the lower concentrations
as in the germination bioassays (Chapter 2). This growth stimulation is an indication
that C. bonariensis plants did in fact produce and release putative allelopathic
compounds, however, the concentrations were not high enough in the pots of the
receiver plants to cause plant growth inhibition, since almost half of the 200ml
applied to pots leached every morning. The second explanation for these results is
the duration of the experiment. Due to the nature of the growth media used in the
experiments, results suggest that there was a need for the experiment to be
conducted for a longer period in order to allow for putative allelopathic compounds to
accumulate to higher (toxic) concentrations in the pots and plants. Therefore
terminating the trial after four weeks was perhaps premature. In addition to the
duration of the experiment, the leaching of the allelochemicals was probably done at
too short intervals. It might have been beneficial to implement leaching treatments on
donor pots on a weekly basis in order for the allelochemical concentration in the
donor plant pots to accumulate. In a leachate pot experiment by Viard-Cretat et al.
(2009) to investigate whether the release of allelochemicals by the dominant tussock
grass (Festuca paniculata) is responsible for its dominance by inhibiting growth of
neighbour grasses in subalpine grasslands, plant species were given enough time (1
year) to exert a chemical influence on the soil medium
3.4 Conclusion
Results from the hydroponic experiment indicate that C. bonariensis roots contain
and release growth inhibitors that are capable of reducing the growth of lettuce.
Although C. bonariensis from Hatfield and those from the Western Cape differ
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morphologically, the degree of phytotoxicity on lettuce plant growth did not vary,
except in the case of root dry mass where the Hatfield population caused
significantly greater reduction in lettuce root growth than the other two populations.
While allelopathy seemed to have played a major role in the results of this
experiment, one must not rule out the possibility of competition. For example, even
though the feeding solution was balanced and changed every week it is still possible
that certain micro- or macro elements might have not become limiting during each
week. It is also possible that C. bonariensis could have been a better competitor for
light due to its growth form. The leachate experiment demonstrated that, as with
many other weed species, the allelopathic potential of C. bonariensis varies with
plant species exposed to the potential allelochemicals and amount of allelochemicals
present. Effects of C. bonariensis leachate on tomato plant growth confirmed that
allelopathy as an interference mechanism is not only harmful (inhibitory) but
apparently can also be beneficial (stimulatory). The observed stimulatory effects of
C. bonariensis at certain leachate concentrations should be investigated further.
Although the scope of this study precluded chemical identification of allelochemicals
involved in the responses of test species, modern molecular and biotechnological
tools allow for in-depth studies on allelopathy, the role of root exudates in
allelopathy, and the linking of such plant attributes with the interfering/invasive ability
of particular plants in both agro- and natural ecosystems.
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CHAPTER 4
REPLACEMENT SERIES APPROACH FOR DETERMINING THE RELATIVE
INTERFERENCE OF CONYZA BONARIENSIS IN RELATION TO LETTUCE AND
TOMATO
4.1 Introduction
Allelopathy is better demonstrated through experiments in which a toxic product is
shown to be released from the putative aggressor, and arrives at the putative victim
in functional concentrations under reasonably natural conditions (Blum, 1995). The
plant with allelopathic potential is referred to as the "donor plant," while the plant in
the surrounding area affected by the allelopathic compounds from the donor plant is
referred to as the "receiver plant." Donor and receiver plants can affect each other
through allelopathy and competition (Muller, 1969).
The relative density between donor and receiver species is believed to be an
important factor in the degree of expression of allelopathy and this has been
suggested as a method to distinguish between allelopathy and resource competition.
Weidenhamer et al. (1989) were among some of the first scientists to demonstrate
that allelopathic interference and resource competition can be distinguished
experimentally by the density-dependent nature of phytotoxic effects, which cause
deviations from predicted yield-density relationships. For monocultures, phytotoxicity
decreases as plant density increases as a result of the dilution of the available toxin
among many plants at high densities, such that each receives a sub-lethal dose. As
the observation of growth reductions at low but not at high densities is inconsistent
with a hypothesis of resource competition, such results constitute strong evidence for
the presence of an inhibitor in soil. An experimental design that demonstrated
allelopathic interference in mixed cultures of two species would be more broadly
applicable (Wu et al., 2002).
Previously researchers have used two different experimental designs, additive and
replacement series, to study the interactive behaviour of components in mixed
stands. In additive series (e.g., Donald 1958), various densities of a second species
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supplement a constant density of an indicator species. Studies using additive series
have normally demonstrated that increasing densities of the second species depress
the yield of the indicator species. In replacement series (e.g., de Wit 1960), a
constant total density of plants is used and the planting density of one species is
proportionately decreased as the planting density of the second species is
increased.
In ecology, replacement series have been used to explore many issues, including
species coexistence, exclusion, co-adaptation, niche differentiation, abundance,
distribution, productivity and diversity (e.g., Aberg et al., 1943; Black, 1958). In
agriculture and forestry, replacement series have regularly been used in studies of
weed-crop associations, and they are the common setting for evaluating yield
advantages in intercrops (Jolliffe, 2000). Experiments that use multiple densities
make it possible to compare monoculture stands, and allow for the determination of
the relative extent of intra-and interspecific competition between the species (Jolliffe
et al., 1984; Santos et al., 1997).
Relative yield total (RYT) and relative yield (RY) are commonly used variables to
calculate the yield of a species in the mixture as a proportion of its yield in
monoculture and thus measures interspecific and intraspecific competition (Santos et
al., 1997; Hector, 2006). An RYT less than one implies that mutual antagonism is
occurring (Harper, 1977) or that not all the resources available to plants are being
used. One explanation for this non-use may be that plants may inhibit the growth of
each other through allelopathy (Putnam and Tang, 1986). The objective of this study
was to assess the allelopathy of C. bonariensis in relation to that of lettuce and
tomato by increasing C. bonariensis plant density, and thus increasing the
concentration of putative compounds with allelopathic potential in the growth
medium.
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4.2 Materials and Methods
Replacement series experiments were conducted in a greenhouse at the Hatfield
experimental farm. The experimental design was completely randomized (Figure
4.1). C. bonariensis plants were collected at the rosette stage on the Hatfield
experimental farm, and were grown in pots (20 cm height x 20 cm diameter) in 4 kg
sterilized field soil (sandy-loam) together with lettuce(Lactuca sativa) and tomato
(Lycopersicon esculentum) seedlings. Lettuce and tomato seedlings were obtained
from Die Tuinhoekie nursery, and the seedlings were in the 2-leaf stage when
transplanted to the pots. Treatments consisted of combinations of six proportions of
C. bonariensis and lettuce and combinations of six proportions of C. bonariensis and
tomato. The experiment was laid out in replacement series as outlined by
Radosevich et al., 1996. Each treatment was replicated five times.
The treatment combinations were arranged in two sets, as follows:
1. 5 C. bonariensis+ 0 lettuce (C. bonariensis monoculture)
2. 4 C. bonariensis+ 1 lettuce
3. 3 C. bonariensis+ 2 lettuce
4. 2 C. bonariensis+ 3 lettuce
5. 1 C. bonariensis+ 4 lettuce
6. 0 C. bonariensis+ 5 lettuce (lettuce monoculture)
1. 5 C. bonariensis+ 0 tomato (C. bonariensis monoculture)
2. 4 C. bonariensis+ 1 tomato
3. 3 C. bonariensis+ 2 tomato
4. 2 C. bonariensis+ 3 tomato
5. 1 C. bonariensis+ 4 tomato
6. 0 C. bonariensis+ 5 tomato (tomato monoculture)
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Pots were surface-watered with 200 ml of Hoagland‘s nutrient solution every second
day throughout the experiment with the purpose of excluding competition for water
and nutrients. The experiment was set up in a glasshouse with natural light and
temperature range of 18 to 28 ºC. Harvesting of the trial was done four weeks after
treatment commenced and dry mass of shoots (all above ground parts) and roots
were measured. To obtain dry mass, plants were divided into shoot and root and put
to dry in a forced air circulation incubator at 60ºC for a period of 72 hours. Then
weighing was conducted; mean dry mass corresponded to the sum of shoot dry
mass plus root dry mass ratio in each proportion. Data were subjected to analysis of
variance and mean separation was done with the least significant difference test of
Tukey at P=0.05. Analysis of variance (ANOVA) was done using the statistical
programme SAS 9.2 (2002).
Figure 4.1 A replacement series experiment to investigate the effect of different
densities of C. bonariensis on plant growth of lettuce
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Relative yield (RY) and relative yield total (RYT) (Radosevich, 1988) were calculated
as:
The relative yield of both the crop and the weed can be calculated as summed to
give the relative yield totals (RYT). The RYT can be used to describe the mutual
interaction that occurs between the species:
1. RYT = 1: this situation implies that each species is making the same demands for
"space" as the other; they are "mutually exclusive" or complementary.
2. RYT > 1: this situation suggests that one or both of the species are less affected
by interspecific interactions than could be predicted from their monoculture
responses; it suggests that they are: (a) making different demands on the same
resources; (b) occupy different niches in time or space; or (c) exhibit some sort of
symbiotic relationship.
3. RYT < 1: this situation occurs when one or both species are more negatively
affected by interspecific competition than would be expected from their pure stand
responses and indicates mutual antagonism. Possible mechanisms that could
explain this interaction are: (a) the action of allelopathic compounds produced by one
or both species, or (b) the loss of the pure stand effect in the mixture.
Traditionally, the RYT concept applies to competition studies only. In our approach
we attempted to eliminate competition in order to make findings that are only
applicable to allelopathic effects.
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4.3 Results and Discussion
4.3.1 Dry mass
Lettuce: Results for dry mass of lettuce grown at different proportions with C.
bonariensis show that there were no significant effects on the growth of the crop
species at all proportions. Even though there is a trend for apparent growth
stimulation of C. bonariensis at proportion 3 lettuce: 2 Conyza, it is not statistically
significant when compared to the control.
Figure 4.2 Dry mass of C. bonariensis and lettuce grown together in a replacement
series at different proportions (Means with same letters do not differ significantly at
P=0.05; ANOVA presented in Appendix C, Table C1)
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Figure 4.3 Root and shoot growth comparison between C. bonariensis and L. sativa
from the replacement series. A: 5 lettuce + 0 C. bonariensis; B: 0 lettuce + 5 C.
bonariensis; C: 4 C. bonariensis +1 lettuce; D: 3 C. bonariensis +2 lettuce; E: 2 C.
bonariensis + 3 lettuce; F: 1 C. bonariensis + 4 lettuce
Allelopathy is usually interspecific; but if the donor and the recipient belong to same
species it becomes intraspecific allelopathy and the term used is autotoxicity.
Therefore, autotoxicity occurs when a plant releases toxic chemical substances into
the environment that inhibit germination and growth of the same plant species
(Miller, 1996). According to Reinhardt et al. (1999) it is probable that autotoxicity may
have a confounding influence when the growth of species grown together is
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compared. It can be seen from Figure 4.3 A and B that there may have autotoxicity
when lettuce and C. bonariensis where grown separately in the controls. Autotoxicity
has been reported for Asteraceae weeds such as Amaranthus palmerii, Helianthus
occidentalis, Parthenium hysterophorus and Plantago lanceolate (Curtis and Cottam,
1950; Newman and Rovira, 1975; Kumari and Kohli, 1987). It is also possible that
lettuce and C. bonariensis in the combinations mentioned above were involved in
intra-species competition. Although competition for water and nutrients were
eliminated by regularly adding a nutrient solution, competition for light and space
could have still taken place.
Tomato: As with lettuce, dry mass for tomato plants grown with C. bonariensis
showed no significant differences when compared to the control. The trend for
apparent growth stimulation of C. bonariensis at all combinations is statistically not
significant.
Figure 4.4 Dry mass of Conyza bonariensis and tomato grown together in a
replacement series at different proportions (Means with same letters do not differ
significantly at P=0.05; ANOVA presented in Appendix C, Table C2)
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Figure 4.5 Root and shoot growth comparison between C. bonariensis and tomato
from the replacement series. A: 5 tomato + 0 C. bonariensis; B: 5 C.bonariensis + 0
tomato; C: 4 C. bonariensis + 1 tomato; D: 3 C. bonariensis + 2 tomato; E: 2 C.
bonariensis + 3 tomato; F: 1 C. bonariensis + 4 tomato
Tomato and C. bonariensis plants in Figure 4.5 A and B suggest that there may have
been autotoxicity involved in the controls of both the donor and test species. C.
bonariensis in Figure 4.5 C and D exhibit apparent autotoxicity with some plants
being smaller than others, just as in the lettuce experiment.
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When comparing the tomato plants in Figure 4.5 F (1 C. bonariensis + 4 tomato) to
those in other plant density combinations of tomato and C. bonariensis, there is
apparent growth stimulation in tomato. This could have been due to the single C.
bonariensis plant in that particular mixture producing such low concentration of
allelochemical(s) that the effect was stimulatory instead of inhibitory. The same
response was observed in Chapter 2, Figure 2.6.In a replacement series study by
Santos et al. (1997), in which tomato plants were grown with yellow and purple
nutsedge, results showed that tomato dry weight per plant increased and dry weight
per plant of nutsedge decreased as their relative proportions decreased in mixture.
4.3.2 Relative yield
Lettuce: At all the combinations of lettuce and C. bonariensis the relative yield of C.
bonariensis was slightly greater than that of lettuce. Up to 3 lettuce: 2 C. bonariensis,
RYT was increasing with increasing lettuce number which means the less C.
bonariensis the more RYT. At 4 Lettuce: 1 C. bonariensis combination, RYT
decreased and relative yield of lettuce was < 1, suggesting that there was an action
of phytotoxins produced by one or both species. This effect was probably due to
lettuce autotoxicity, considering that there was more lettuce plant in the pot.
Figure 4.6 Relative yields (RY) of lettuce and C. bonariensis and relative yield total
(RYT) four weeks after transplanting under different densities and proportions
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Tomato: For all the combinations of tomato and C. bonariensis, the RY of tomato
was greater than that of C. bonariensis. According to Bianchi et al. (2006), generally,
replacement experiments demonstrate that the crop is more competitive than the
weed species, since the effect of the weeds in crops is not due to their higher
competitive ability, but to the degree of infestation. However, their research did not
consider allelopathy. RYT in all the combinations was > 1 which probably implies that
both species were less affected by interspecific interactions than in their respective
monocultures.
Figure 4.7 Relative yields (RY) of tomato and C. bonariensis and relative yield total
(RYT) four weeks after transplanting under different densities and proportions
4.4 Conclusions
According to the results of this study lettuce and tomato possess phytotoxic ability
equivalent to that of C. bonariensis in relation to total dry mass, given that there were
no significant differences in plant mass between the various combinations. It is
important to consider that the inhibition of lettuce and tomato germination assigned
to allelochemicals produced by C. bonariensis and growth observed in previous
studies (Chapter 2 and 3) is not ostensible in the results of this study because of
methodology, choice of growth media and competition.
The methodology in this chapter may have attempted to eliminate competition
resulting from nutrient and water stress; however, allelopathy in soil is a complicated
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phenomenon that is governed not only by the physiochemical properties but also by
the soil organic matter and microorganisms. It has been suggested that the
concentration of an allelochemical in soil water is a dominant factor directly
determining the phytotoxic activity in the soil (Kobayashi, 2004), and that the
concentration is controlled by soil factors that affect the behavior of adsorption,
desorption and degradation in soil. Therefore, it is probable that since the
allelochemicals in the plant extract and hydroponic experiments were in water or
similar media, and therefore not affected by soil factors, the concentration of
allelochemicals was therefore not affected and reached the receiver plants in
relatively high doses. Poor correspondences between bioassays and field studies
have often been found. Belz et al. (2009) demonstrated that although much research
has been done to study the allelopathic potential of P. hysterophorus, its
invasiveness could not be attributed to the plant metabolite parthenin, when its
persistence and phytotoxicity in soil was studied.
Growth stage of receiver plants is also one of many factors that affect phytotoxicity of
allelochemicals. Allelopathy is usually more pronounced at seed germination and
early seedling development stages. Plants used in this study were more matured
than seed/seedlings used in the seed germination bioassay, thus making them less
susceptible to the putative allelochemicals. Finally, in the germination bioassay it
was found that the leaves of C. bonariensis contained more phototoxic compounds
when compared to the roots, but in the present study receiver plants were only
exposed to compounds released by roots. In conclusion, although C. bonariensis
exhibited statistically significant and often dramatic phytotoxicity on lettuce and
tomato in previous experiments, this study reveals that there may be other aspects
connected to the allelopathic potential of C.bonariensis in the field.
Finally, the third aspect to consider in the almost non-existent allelopathic effect of C.
bonariensis on the crop species is competition for light. Setting up an experiment in a
greenhouse with natural light hardly eliminates competition if the leaves of the plants
start to grow over one another; particularly in the case of lettuce where large leaves
may compete for space and space and light.
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CHAPTER 5
GENERAL DISCUSSION AND CONCLUSION
Much research on Conyza bonariensis has been mainly directed towards its
resistance to herbicides, while literature concerning its interference mechanisms and
possible allelopathic interference with crop species is extremely limited. The weed
originates from South America and was first reported to occur in South Africa in May
1895 in Franschoek (De Wet, 2005). This weed is currently invading cultivated and
non-cultivated lands, gardens, roadsides and waste places, with infestations of one
or more species in every province (Danin, 1990; Botha, 2001). Due to the
importance of this weed, it would be of great value to know and understand the
mechanisms by which C. bonariensis interferes with crops. This investigation on the
allelopathic potential of C. bonariensis was done to evaluate whether this alien
invader has the capacity to interfere with other species in this way.
5.1 Allelopathic influence of Conyza bonariensis on lettuce and tomato seed
germination and early seedling development
The study presented in Chapter 2 was aimed at investigating the allelopathic effects
of aqueous extracts of C. bonariensis on seed germination and seedling growth of
two test species; and to verify whether the compounds influencing germination were
polar or non-polar in nature, and if they have different impacts on seed germination
and seedling growth of the test species. This study revealed the presence of
allelopathic substances in leaves and roots of C. bonariensis. Germination and early
seedling growth of both lettuce and tomato were inhibited by aqueous infusions and
by hexane extracts. The possible confounding effects of osmotic potential of extracts
were negated. From the results obtained it is clear that putative allelochemicals
contained in C. bonariensis leaves are more potent than those in the roots. Since
compounds extracted by hexane would not be water-soluble, the lower potency
observed with these extracts could have been due to poor or zero absorption by
seed/seedlings or the fact that the inhibitory compounds present in C. bonariensis
are polar in nature .
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5.2 Assessment of the allelopathic potential of Conyza bonariensis root
exudates
Albeit laboratory bioassays are valuable when investigating the allelopathic potential
of plant extracts, they are not sufficient in concluding the presence of allelopathic
compounds under natural conditions. The use of plant extracts is often criticized
because it is too far removed from what occurs in nature. This experiment was
conducted with two main objectives: the first was to investigate whether C.
bonariensis contains and releases, through its roots, chemicals with allelopathic
potential by growing it together with test species in a nutrient solution, and using
plant growth as a measure of effect. The second objective was to determine if
leachate from C. bonariensis affected the growth of test species exposed to different
leachate concentrations. In the hydroponic experiment three populations of C.
bonariensis were used, one from Pretoria and two from the Western Cape. Lettuce
shoot and root growth was significantly reduced by all three populations of C.
bonariensis. Generally, there were no significant differences in the degree of
inhibition caused by the three biotypes on the growth of lettuce, except in the case of
root dry mass results, where the Pretoria population caused significantly greater
reduction in lettuce root growth than the other two populations. These findings
suggest that C. bonariensis produces and releases allelochemicals into the
environment from its roots, at least. In the leachate experiment there was no growth
inhibition observed for both test species. However, there was apparent growth
stimulation of tomato roots at the highest concentration. This stimulatory effect of C.
bonariensis leachate should be investigated further, and if such findings would lend
support, the weed or extracts prepared from it could conceivably be used as a
growth stimulator on responsive crops.
5.3Replacement series approach for determining the relative interference of
Conyza bonariensis in relation to lettuce and tomato
In order to hamper plant growth, allelochemicals must accumulate and persist at
phytotoxic levels in soil (Jilani et al., 2008). Replacement series experiments in
Chapter 4 were conducted under greenhouse conditions to assess the allelopathy of
Conyza bonariensis in relation to that of lettuce and tomato by increasing C.
bonariensis plant density, thus increasing the concentration of putative compounds
with allelopathic potential in the growth medium. These experiments represented an
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environment that is closer to natural field conditions than the bioassay approach.
Results for dry mass of lettuce and tomato grown at different proportions with C.
bonariensis showed that there were no significant effects on the growth of the crop
species at all proportions. Furthermore, except for the proportion 4:1 lettuce: C.
bonariensis, RYT was > 1 at all the combinations, which probably implies that both
crop species and C. bonariensis were less affected by interspecific interactions than
in their respective monocultures. The difference in results obtained in Chapter 4 in
relation to those in Chapter 2 and 3 are attributed to methodology and growth media.
Since plants in Chapter 4 were grown in natural soil, it is highly probable that, unlike
in the plant extract and hydroponic experiments where water media were used,
allelochemicals were not absorbed by receiver plants in lethal dosages. Growth
stage of receiver plant and plant organ of donor plant are the other two factors
suspected to have restricted the phytotoxicity of allelochemicals in this experiment.
In the preceding bioassay studies, the acceptor species were in the seed/seedling
growth stages when it was concluded that the leaves of C. bonariensis contained
allelochemicals of higher potency than the roots. We propose that allelochemicals in
the present experiment were either adsorbed on soil colloids and/or were
metabolized by soil microorganisms. This theory, however, needs to be
substantiated with further investigations.
.
5.4 Conclusions and recommendations
Current findings suggest that mature C. bonariensis plants can detrimentally affect
the germination and early seedling growth of other plants, e.g., the crop, and that the
relative maturity of crop and weed determines the nature and intensity of the
allelopathic interaction. For example, if crop seed are sown into an environment
where there are either live C. bonariensis plants or dead weed material that was
either incorporated into the soil or present on the soil surface, it would constitute a
risk of allelopathic effect on the crop. It is suggested that this investigation provides
strong evidence that C. bonariensis has significant allelopathic potential, as shown
by inhibition of seed germination and early seedling development of lettuce and
tomato. C. bonariensis from the Western Cape and Gauteng provinces (populations
separated by more than 1,000 km) showed this potential. The highest allelopathic
potential (potency) may be found in the leaves of the plant, with lower potency
occurring in the roots. A possible explanation for this difference in potency between
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plant organs could be found in the ecological strategy of this weed. Perhaps this
weed species relies on allelopathy exerted by its above-ground material, which
logically could become relevant in the case of high infestation levels. For C.
bonariensis to have a direct phytotoxic effect on other plants its allelochemicals must
be available in the soil for plant uptake at sufficiently high concentrations.
Further research should be performed to identify the active compounds involved in
the allelopathy of C. bonariensis and their fate and persistence in soil. Shoots and
roots of tomato were significantly stimulated in the germination bioassay and
leachate experiment, respectively. In the replacement series, although not
significant, there was apparent growth stimulation of tomato plants when grown with
a single C.bonariensis plant, which corresponds with growth stimulation of tomato
shoots at low extract concentrations in the germination bioassay. The apparent
stimulatory effects observed can be attributed to the phenomenon of hormesis,
where a particular allelochemical can stimulate plant growth at sub-lethal
concentrations, and inhibit growth at higher concentrations. In order to verify the
practical relevance of knowledge contributed by this study, future work should also
involve field experiments. However, the study of allelopathy in field trials will pose
further challenges, the main one being the need to separate competition and
allelopathy. Until such time as further research yields better understanding of the
practical relevance of the allelopathic potential of C. bonariensis, crop producers and
weed management practitioners should recognize that this important weed has the
ability to interfere with the growth and development of a crop through two
mechanisms, competition plus allelopathy. In essence, what this boils down to is that
weeds should not be allowed to attain telling numbers and/or mass on crop fields,
and hence, weeds must be controlled at an early growth stage, in accordance with
recommendations appearing on herbicide product labels.
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SUMMARY
ALLELOPATHIC POTENTIAL OF CONYZA BONARIENSIS
Conyza bonariensis is widespread in the world, and has become a common weed of
cultivated and non-cultivated lands, gardens, roadsides and waste places in South
Africa over the past century. Since then the weed has not only naturalized itself in
many parts of the country but has spread in an alarming rate, and exploded into
aggressive herbicide resistant populations. Despite the importance of this weed in
agroecosystems little is known about the mechanisms by which this weed competes
with crops. The possibility that C. bonariensis populations in South Africa might
possess allelopathic compounds was researched in this study.
Initial investigations focussed on the allelopathic interference of C. bonariensis on
lettuce and tomato seed germination and early seedling development under
laboratory conditions. Crude extracts for preparation of test solutions were obtained
using two solvents (water and hexane) to verify whether the compounds influencing
germination were polar or non-polar in nature. To refine the bioassay technique
attempts were made to eliminate, or at least reduce, possible confounding factors
such as osmotic inhibition, pathogenic microorganisms and phytotoxic residues of
organic solvents used for extraction. In all bioassays, germination percentage, root
and shoot growth of both test species were inhibited following exposure to aqueous
and hexane extracts of roots and leaves of the weed. Evaluation of the results
obtained suggested that the leaves of C. bonariensis are the main site of
allelochemicals. These results show that incorporation of live or dead material of the
weed into the soil could negatively affect the establishment of crop species.
The second investigation was divided into two parts. Firstly, the ability of C.
bonariensis to release putative allelochemicals through its roots was studied using
three provenances of the weed (one from Pretoria in Hatfield, and two from the
Western Cape), and the second study‘s was aimed to determine if leachate from C.
bonariensis affected the growth of test species exposed to different leachate
concentrations. For both experiments, plants were grown in a greenhouse on the
Experimental Farm at the University of Pretoria. In the first experiment, in which the
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weed and test species (lettuce) were grown together hydroponically, highly
significant differences were observed in the growth of lettuce plants grown with C.
bonariensis when compared to the control. This growth reduction in lettuce plants
may indicate that even though C. bonariensis may have the highest content in its
leaves, the roots may also release putative allelochemicals into the environment.
Another interesting feature in this study was that the Hatfield population caused
significantly greater reduction in lettuce root growth than the other two populations.
In the second experiment growth inhibition was not observed for lettuce and tomato
plants treated with C. bonariensis leachate supplied at different concentrations.
However, there was apparent growth stimulation of tomato roots at the highest
concentration.
In the third investigation a greenhouse replacement series experiment was
conducted for determining the relative interference of C. bonariensis in relation to
lettuce and tomato. The use of soil in this investigation was to narrow the gap
between laboratory and field conditions. Results for dry mass of lettuce and tomato
grown at different proportions with C. bonariensis showed that there were no
significant effects on the growth of the crop species at all proportions. Relative yield
calculations at all combination imply that both crop species and C. bonariensis were
less affected by interspecific interactions than in their respective monocultures. The
contrasting results obtained in this study were attributed to methodology.
The results of the above mentioned studies suggest that C. bonariensis possesses
allelopathic potential and that the weed could have significant debilitating effects on
agriculture and natural ecosystems. The possible main site of allelochemicals could
be the leaves. Results reported here are from laboratory bioassays and pot
experiments, further research should be extended to the field using more crop
species for studying weed-crop interactions.
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99
© University of Pretoria
APPENDIX A: Chapter 2
Bioassays to determine Conyza bonariensis’ allelopathic potential.
Table A1. Abbreviated ANOVA table for germination bioassays of lettuce
exposed to C. bonariensis leaf infusions (Figure 2.1)
Source
DF
Sum of Squares
Mean Square
F Value
Corrected Total
99
86811.00000
Plantpart
1
Concentration
169.00000
169.00000
0.83
4
55016.00000
13754.00000
67.83
<.0001
Plantpart*Concentration 4
13376.00000
3344.00000
16.49
<.0001
Error
18250.00000
202.77778
90
Pr > F
0.3637
Table A2. Abbreviated ANOVA table for germination bioassays of lettuce root
(radicle) growth exposed to C. bonariensis leaf and root infusions (Figure 2.2)
Source
DF
Corrected Total
99
10882.16000
Plantpart
1
108.160000
Concentration
4
Plantpart*Concentration 4
Error
90
Sum of Squares
F Value
Pr > F
108.160000
10.42
0.0017
9606.560000
2401.640000
231.27
<.0001
232.840000
58.210000
5.61
934.60000
Mean Square
0.0004
10.38444
Table A3. Abbreviated ANOVA table for germination bioassays of lettuce shoot
growth exposed to C. bonariensis leaf and root infusions (Figure 2.3)
Source
DF
Corrected Total
99
6144.910000
Plantpart
1
778.410000
778.410000
57.18
<.0001
Concentration
4
3828.660000
957.165000
70.31
<.0001
312.540000
78.135000
5.74
1225.300000
13.614444
Plantpart*Concentration 4
Error
90
Sum of Squares
Mean Square
F Value
Pr > F
0.0004
100
© University of Pretoria
Table A4. Abbreviated ANOVA table for germination bioassays of tomato
exposed to C. bonariensis leaf and root infusions (Figure 2.4)
Source
DF
Sum of Squares
Corrected Total
99
116811.0000
Plantpart
1
5929.00000
Concentration
4
F Value
Pr > F
5929.00000
41.14
<.0001
82316.00000
20579.00000
142.80
<.0001
Plantpart*Concentration 4
15596.00000
3899.00000
27.06
<.0001
Error
12970.0000
90
Mean Square
144.1111
Table A5. Abbreviated ANOVA table for germination bioassays of tomato root
(radicle) growth exposed to C. bonariensis leaf and root infusions (Figure 2.5)
Source
DF
Sum of Squares
Corrected Total
99
42952.11000
Plantpart
1
1391.29000
1391.29000
41.89
Concentration
4
37179.26000
9294.81500
279.84
Plantpart*Concentration 4
1392.26000
348.06500
10.48
Error
2989.30000
33.21444
90
Mean Square
F Value
Pr > F
<.0001
<.0001
<.0001
Table A6. Abbreviated ANOVA table for germination bioassays of tomato shoot
growth exposed to C. bonariensis leaf and root infusions (Figure 2.6)
Source
DF
Corrected Total
99
17034.75000
Plantpart
1
26.01000
Concentration
4
Plantpart*Concentration 4
Error
90
Sum of Squares
Mean Square
F Value
26.01000
1.93
14409.40000
3602.35000
267.21
1386.04000
346.51000
25.70
1213.30000
Pr > F
0.1683
<.0001
<.0001
13.48111
101
© University of Pretoria
Table A7. Abbreviated ANOVA table for germination bioassays of lettuce
exposed to C. bonariensis hexane leaf and root extract (Figure 2.7)
Source
DF
Corrected Total
99
Sum of Squares
Mean Square
F Value
Pr > F
19136.00000
Plantpart
1
4.000000
4.000000
0.03
0.8624
Concentration
4
4366.000000
1091.500000
8.24
<.0001
Plantpart*Concentration 4
2846.000000
711.500000
5.37
0.0006
Error
11920.00000
132.44444
90
Table A8. Abbreviated ANOVA table for germination bioassays of lettuce roots
(radicles) exposed to C. bonariensis hexane leaf and root extract (Figure 2.8)
Source
DF
Sum of Squares
Mean Square
F Value
Corrected Total
99
15260.75000
Plantpart
1
Concentration
5730.490000
5730.490000
247.02
<.0001
4
4875.100000
1218.775000
52.54
<.0001
Plantpart*Concentration 4
2567.260000
641.815000
Error
2087.90000
23.19889
90
27.67
Pr > F
<.0001
Table A9. Abbreviated ANOVA table for germination bioassays of lettuce shoot
growth exposed to C. bonariensis hexane root extract (Figure 2.9)
Source
DF
Sum of Squares
Corrected Total
99
1868.510000
Plantpart
1
Concentration
4
Plantpart*Concentration 4
Error
90
Mean Square
F Value
272.250000
272.250000
53.86
<.0001
1035.160000
258.790000
51.20
<.0001
26.550000
5.25
106.200000
454.900000
Pr > F
0.0008
5.054444
102
© University of Pretoria
Table A10. Abbreviated ANOVA table for germination bioassays of tomato
exposed to C. bonariensis hexane leaf extract (Figure 2.10)
Source
DF
Sum of Squares
Mean Square
F Value
Corrected Total
99
19804.00000
Plantpart
1
484.000000
484.000000
4.46
Concentration
4
8494.000000
2123.500000
19.58
Plantpart*Concentration 4
1066.000000
266.500000
Error
90
9760.00000
2.46
Pr > F
0.0374
<.0001
0.0512
108.44444
Table A11. Abbreviated ANOVA table for germination bioassays of tomato root
(radicle) growth exposed to C. bonariensis hexane leaf and root extract (Figure
2.11)
Source
DF
Sum of Squares
Corrected Total
99
30114.75000
Mean Square
F Value
Plantpart
1
506.25000
506.25000
15.86
Concentration
4
26037.70000
6509.42500
203.96
698.50000
174.62500
5.47
Plantpart*Concentration 4
Error
90
2872.30000
Pr > F
0.0001
<.0001
0.0005
31.91444
Table A12. Abbreviated ANOVA table for germination bioassays of tomato
shoot growth exposed to C. bonariensis hexane root extract (Figure 2.12)
Source
DF
Sum of Squares
Corrected Total
99
2056.360000
Mean Square
F Value
Pr > F
Plantpart
1
163.8400000
163.8400000
18.74
<.0001
Concentration
4
860.4600000
215.1150000
24.61
<.0001
Plantpart*Concentration 4
245.2600000
61.3150000
Error
90
786.800000
7.01
<.0001
8.742222
103
© University of Pretoria
APPENDIX B: Chapter 3
Assessment of the allelopathic potential of Conyza bonariensis root exudates
Table B1. Abbreviated ANOVA table for leaf fresh mass of lettuce grown
hydroponically with C. bonariensis plants collected on Hatfield experimental
farm (Figure 3.5)
Source
DF
Sum of Squares
Mean Square
F Value
72.47
Corrected Total
19
9037.877280
Treatment
1
7239.773520
7239.773520
Error
18
1798.103760
99.894653
Pr > F
<.0001
Table B2. Abbreviated ANOVA table for root fresh mass of lettuce grown
hydroponically with C. bonariensis plants collected on Hatfield experimental
farm (Figure 3.5)
Source
DF
Sum of Squares
Mean Square
F Value
56.80
Corrected Total
19
844.1960550
Treatment
1
641.0516450
641.0516450
Error
18
203.1444100
11.2858006
Pr > F
<.0001
Table B3. Abbreviated ANOVA table for leaf dry mass of lettuce grown
hydroponically with C. bonariensis plants collected on Hatfield experimental
farm (Figure 3.8)
Source
DF
Sum of Squares
Corrected Total
19
61.66678000
1
18
Treatment
Error
Mean Square
F Value
39.81842000
39.81842000
32.80
21.84836000
1.21379778
Pr > F
<.0001
104
© University of Pretoria
Table B4. Abbreviated ANOVA table for root dry mass of lettuce grown
hydroponically with C. bonariensis plants collected on Hatfield experimental
farm (Figure 3.8)
Source
DF Sum of Squares Mean Square F Value Pr > F
Corrected Total
19
9.38125500
Treatment
1
5.95140500
5.95140500
Error
18
3.42985000
0.19054722
31.23
<.0001
Table B5. Abbreviated ANOVA table for leaf fresh mass of lettuce grown
hydroponically with two Western Cape provenances of C. bonariensis (Figure
3.9)
Source
DF
Corrected Total
29
Treatment
2
Error
27
Sum of Squares
Mean Square
F Value
3807.489330
25.90
Pr > F
11584.02212
7614.978660
3969.04346
<.0001
147.00161
Table B6. Abbreviated ANOVA table for root fresh mass of lettuce grown
hydroponically with two Western Cape provenances of C. bonariensis(Figure
3.9)
Source
DF
Sum of Squares
Mean Square
Corrected Total
29
788.7474667
Treatment
2
525.9911267
262.9955633
Error
27
262.7563400
9.7317163
F Value
Pr > F
27.02
<.0001
Table B7. Abbreviated ANOVA table for leaf dry mass of lettuce grown
hydroponically with two Western Cape provenances of C. bonariensis (Figure
3.10)
Source
DF
Sum of Squares
Corrected Total
29
60.57773667
2
27
Treatment
Error
Mean Square
F Value
29.06312667
14.53156333
12.45
31.51461000
1.16720778
Pr > F
0.0001
105
© University of Pretoria
Table B8. Abbreviated ANOVA table for root dry mass of lettuce grown
hydroponically with two Western Cape provenances of C. bonariensis (Figure
3.10)
Source
DF
Sum of Squares
Corrected Total
29
8.26692417
Treatment
2
3.83442167
Error
27
4.43250250
Mean Square
F Value
1.91721083
11.68
Pr > F
0.0002
0.16416676
Table B9. Abbreviated ANOVA table for leaf fresh mass of lettuce that was
exposed to C. bonariensis leachate (Figure 3.11)
Source
DF
Corrected Total
24
Treatment
4
Error
20
Sum of Squares
Mean Square
F Value
Pr > F
7166.788296
1803.072976
5363.715320
450.768244
1.68
0.1938
268.185766
Table B10. Abbreviated ANOVA table for root fresh mass of lettuce that was
exposed to C. bonariensis leachate(Figure 3.11)
Source
DF
Sum of Squares
Corrected Total
24
120.2069440
4
48.10766400
20
72.0992800
Treatment
Error
Mean Square
12.02691600
F Value
3.34
Pr > F
0.0301
3.6049640
Table B11. Abbreviated ANOVA table for leaf dry mass of lettuce that was
exposed to C. bonariensis leachate(Figure 3.12)
Source
DF
Corrected Total
24
Treatment
Error
Sum of Squares
Mean Square
F Value
1.58
Pr > F
24.75205600
4
5.93213600
1.48303400
20
18.81992000
0.94099600
0.2193
106
© University of Pretoria
Table B12. Abbreviated ANOVA table for leaf dry mass of lettuce that was
exposed to C. bonariensis leachate (Figure 3.12)
Source
DF
Sum of Squares
Corrected Total
24
4.91153400
4
1.70381400
0.42595350
20
3.20772000
0.16038600
Treatment
Error
Mean Square
F Value
2.66
Pr > F
0.0631
Table B13. Abbreviated ANOVA table for leaf fresh mass of tomato that was
exposed to C. bonariensis leachate (Figure 3.13)
Source
DF
Corrected Total
Treatment
Error
Sum of Squares
Mean Square
24
623.0834000
4
192.2481200
48.0620300
20
430.8352800
21.5417640
F Value
2.23
Pr > F
0.1021
Table B14. Abbreviated ANOVA table for root fresh mass of tomatothat was
exposed to C. bonariensis leachate (Figure 3.13)
Source
DF
Sum of Squares
Corrected Total
24
528.3116160
Treatment
4
Error
20
272.7044560
255.6071600
Mean Square
F Value
68.1761140
5.33
Pr > F
0.0043
12.7803580
Table B15. Abbreviated ANOVA table for leaf dry mass of tomato that was
exposed to C. bonariensis leachate (Figure 3.15)
Source
Corrected Total
DF
Sum of Squares
24
Treatment
4
Error
20
Mean Square
F Value
0.62135600
1.94
Pr > F
8.89534400
2.48542400
6.40992000
0.1432
0.32049600
107
© University of Pretoria
Table B15. Abbreviated ANOVA table for root dry mass of tomato that was
exposed to C. bonariensis leachate (Figure 3.15)
Source
DF
Sum of Squares
Corrected Total
24
4.74825600
4
1.72725600
0.43181400
20
3.02100000
0.15105000
Treatment
Error
Mean Square
2.86
F Value
Pr > F
0.0504
108
© University of Pretoria
APPENDIX C: Chapter 4
The role of allelopathy in Conyza bonariensis inhibition of lettuce and tomato
Table C1. Abbreviated ANOVA table for Dry mass of C. bonariensis and lettuce
grown together in a replacement series at different proportions (Figure 4.4)
Source
DF
Squares
Corrected Total
49
Mean Square
F Value
Pr > F
19.45036800
Species
1
0.01620000
0.01620000
0.04
0.8370
Combinations
4
3.18890800
0.79722700
2.11
0.0973
Species*Combinations
4
1.14182000
0.28545500
0.76
0.5601
Error
40
15.10344000
0.37758600
Table C2. Abbreviated ANOVA table for Dry mass of C. bonariensis and tomato
grown together in a replacement series at different proportions (Figure 4.5)
Source
DF
Corrected Total
Squares
Mean Square
49
F Value
Pr > F
54.97751808
Species
1
4.18414592
4.18414592
4.26
0.0455
Combinations
4
9.03324768
2.25831192
2.30
0.0754
Species*Combinations
4
2.50015168
0.62503792
0.64
0.6393
40
39.25997280
0.98149932
Error
109
© University of Pretoria